Two distinct colonic H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H(+)-K(+)-ATPase alpha-subunit (HKc alpha) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H(+)-K(+)-ATPase activity in apical membranes prepared from surface and crypt cells and K(+)-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H(+)-K(+)-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K(+)-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K(+)-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKc alpha encodes the colonic ouabain-insensitive H(+)-K(+)-ATPase and that a colonic ouabain-sensitive H(+)-K(+)-ATPase isoform is present in colonic crypts and remains to be cloned and identified.