The bile salt export pump (Bsep), a member of the ATP-binding cassette superfamily of transporters, mediates the ATP-dependent canalicular secretion of bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein in Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation sites for protein kinase A, protein kinase C (PKC) and Ca(2+)-calmodulin dependent kinase II, we have also tested whether mBsep undergoes phosphorylation. MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than control membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinity and is the most potent inducer of ATPase activity. Cyclosporin A, glibenclamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt-stimulated ATPase activity. MBsep exists as a phospho-protein when expressed in Sf9 cells and the immunoprecipitated mBsep complex is a substrate for the catalytic subunit of PKC. When mBsep and the alpha-isoform of mouse PKC are co-expressed in Sf9 cells, a ninefold stimulation of phosphorylation occurs. This is further increased to 18-fold after activation by phorbol ester. Given that bile salts activate selected PKC isoforms in hepatocytes, including the alpha isoform, the phosphorylation of mBsep by PKCalpha may represent a point of regulation for this transporter that is mediated by its own substrate.