We have shown that bile acid efflux and ecto-ATPase activities are two distinct properties of a single rat liver hepatocyte canalicular membrane protein (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter, D. H. (1993) J. Biol. Chem. 268, 2083-2091). Bile acid efflux in COS cells transfected with this rat hepatocyte canalicular bile acid transport/ectoATPase cDNA is stimulated by ATP and inhibited by nonhydrolyzable ATP analogs. In this study, we depleted transfected COS cells of ATP to examine whether bile acid efflux mediated by this transporter was dependent on ATP or just stimulated by ATP. We also used mutagenesis of an ATPase consensus sequence in the ectoplasmic domain to examine the relationship of ATPase activity to bile acid efflux mediated by the same polypeptide. The results indicate that bile acid transport is abrogated by ATP depletion and reconstituted by exogenous ATP in a concentration-dependent and saturable manner. Introduction of mutations at amino acids Gly97 and Arg98 in the ATPase consensus sequence abrogated ATPase activity but did not affect synthesis or cell surface delivery of the transporter and did not affect its bile acid transport activity. Taken together, the data indicate that bile acid efflux mediated by the rat hepatocyte canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ATPase activity. The data, therefore, imply that 1) ATP affects its bile acid transport activity through an entirely distinct mechanism; and 2) if there is any functional relationship between the ecto-ATPase and bile acid transport properties, it is mediated indirectly through regulation of net ATP concentrations in the canalicular space by the ecto-ATPase.