Previous studies using isolated tissues suggest that the colonic H, K-ATPase (cHKA), expressed in the colon and kidney, plays an important role in K+ conservation. To test the role of this pump in K+ homeostasis in vivo, we generated a cHKA-deficient mouse and analyzed its ability to retain K+ when fed a control or K+-free diet. When maintained on a control diet, homozygous mutant (cHKA-/-) mice exhibited no deficit in K+ homeostasis compared to wild-type (cHKA+/+ greater, similar mice. Although fecal K+ excretion in cHKA-/- mice was double that of cHKA+/+ mice, fecal K+ losses were low compared with urinary K+ excretion, which was similar in both groups. When maintained on a K+-free diet for 18 d, urinary K+ excretion dropped over 100-fold, and to similar levels, in both cHKA-/- and cHKA+/+ mice; fecal K+ excretion was reduced in both groups, but losses were fourfold greater in cHKA-/- than in cHKA+/+ mice. Because of the excess loss of K+ in the colon, cHKA-/- mice exhibited lower plasma and muscle K+ than cHKA+/+ mice. In addition, cHKA-/- mice lost twice as much body weight as cHKA+/+ mice. These results demonstrate that, during K+ deprivation, cHKA plays a critical role in the maintenance of K+ homeostasis in vivo.