- Marshal Moo Montana: Leader of the C.O.W.-Boys and marshall of Moo Mesa. Courageous and quick on his hooves, Moo Montana "battles the bad guys and makes the West a safer place to graze". Lives by the Code of the West, which he seems to make up as he goes along (episode "The Big Cow Wow"). His horse's name is Cyclone.
- The Dakota Dude: The soft-spoken muscle of Montana's posse, Dakota possesses a cool temperament and rarely gets mad, even in near-death experiences (episode 12: Dances with Bulls). Dakota agreed to marry Cowlamity Kate in Wedding Bull Blues to save her father's inheritance. His horse's name in Rebel and he's scare of heights.
- The Cowlorado Kid: The youngest of the group and a self-proclaimed ladies' man with a good singing voice, regardless of his skill with the lasso and guitar. Cowlorado isn't a deputy yet. In Stolen on the River, he tries to prove he's worthy of being a deputy by catching Five Card Cud only to get in trouble and be rescued by Dakota and Moo.
- Mayor Oscar Bulloney: The greedy and corrupt mayor of Cowtown in Moo Mesa, Bulloney rigs elections (episode 9: Stolen on the River) and makes taxes so high that the Masked Bull compares it to stealing. He also serves as Cowtown's crooked Justice of the Peace and Bank President.
- Sheriff Terrorbull: Selected by the corrupt Bulloney, Terrorbull uses his sheriff's badge to conceal his evil intentions. Was forced to leave Cowtown after losing a bet to Moo in No Face To Hide. Subsequently became sheriff of the remote town of Lonesome Gulch, where he continued his unlawful exploits.
- Lily Bovine: bartender, former showgirl and owner of the local saloon, The Tumbleweed. Also Moo Montana's love interest. Her best friend is Cowlamity Kate.
- Cody Calf: Nicknamed "Calf-Pint" by Moo and his posse, he idolizes the marshall and hopes to be a lawcow himself when he grows up. He is apparently related to Lily Bovine in some way and lives with her, though he does not appear to be her son; like everyone else, he refers to her as "Miss Lily". Though well-meaning, he often gets himself into serious trouble trying to "help" the lawcows, but has been a useful asset on several occasions.
- Cowlamity Kate Cudster: Tomboyish rancher and operator of the highly profitable Golden Cud Mine, and Dakota's love interest. She's as hard-working and hard-riding as any bull, and has enough skill with a lasso to put Cowlorado to shame. She returns Dakota's romantic feelings; she gave him the hat he wears and nearly married him in Wedding Bull Blues.
- Saddle Sore: A scorpion, and one of Sheriff Terrorbull's comically inept henchmen. He is slightly smarter and braver than Boot Hill Buzzard, but only just.
- Boot Hill Buzzard: Sheriff Terrorbull's other comically inept henchman. As the more dim-witted of the two, he is often saddled with more than his fair share of the grunt work, especially if the job involves something embarrassing or unappealing (such as cross-dressing).
- J.R.: An Indian buffalo who occasionally aids Moo, Dakota, and Cowlorado if the situation needs it. He tends to ramble about the scientific principles of his inventions, which the cowboys don't want to listen to.
- Marshal Moo Montana Pat Fraley
- The Dakota Dude & Saddle Sore - Jim Cummings
- The Cowlorado Kid - Jeff Bennett
- Mayor Oscar Bulloney - Michael Greer
- Sheriff Terrorbull - Joe Piscopo
- Lily Bovine - Charity James
- Cody Calf - Troy Davidson
- Cowlamity Kate Cudster - Kay Lenz
- Boot Hill Buzzard - Danny Mann
- J.R. - Michael Horse
Archie Comics released both a mini-series and regular series of Wild West C.O.W.-Boys of Moo Mesa comic books. The first issue of the mini-series came with an exclusive pull-out wanted poster available only through the direct sales market. Three issues of both the mini-series (December 1992, January and February 1993) and the regular series (March, May and July 1993) were released.
The C.O.W.-Boys made their TMNT universe debut when they teamed up with the turtles in issue #21 of Tales of the TMNT. The turtles aided the C.O.W.-Boys in stopping Sheriff Terrorbull from stealing a magic crystal shard.
Moo Montana and his deputies returned in issue #32 of the Tales of the TMNT. This time the horned heroes come up against Savanti Romero and the Eye of Aga-Moo-Tu. The story takes place in the astral realm of Bov-gamesh and introduces the character Tsou-T'an-Jin an ancient sorceress tortoise that lives on Moo Mesa.
The C.O.W.-Boys make their third appearance with the ninja turtles in issue #52 of Tales of the TMNT. The story concluded in Tales of the TMNT #58 printed in May 2009.
It is rumored that there may be an unreleased mini series written by Tristan Jones.