The presence and distribution of gastric-type H(+)-K(+)-ATPase were investigated in the rat colon using a monoclonal antibody raised against hog gastric H(+)-K(+)-ATPase. Rat stomach was used as positive control. Rat kidney and ileum, in both of which H(+)-K(+)-ATPase has been reported in the past, were also studied. In stomach, very strong staining was found confined to the parietal cells, and a strong band at M(r) approximately 94 kDa on the immunoblots. In colon a moderate staining was found in the supranuclear region of the epithelial cells, with similar intensity and distribution of staining of the surface and deep mucosa of the crypts, throughout the length of the colon. Another monoclonal antibody, specific to the 31 kDa subunit of H(+)-ATPase, used as a negative control, or omission of the primary antibody, resulted in lack of any staining in either colon or stomach. On immunoblots of homogenates of colonic mucosa, no specific band could be identified, either due to very low expression of the H(+)-K(+)-ATPase or loss of antigenicity of the epitope during the processing steps. No positive staining was observed in rat kidney and ileum, suggesting that they contain isoforms that are structurally different.