Fluorescence image analysis of isolated rat hepatocyte couplets, which retain a bile canaliculus between them, has shown the presence of transport systems for the bile acid and non-bile acid organic anion in the canalicular membrane. The cells also transported Fura-2 and BCECF, which are fluorescent indicators of intracellular Ca 2+ H+ concentrations, respectively, into the canaliculus. Both Fura-2 and BCECF transports were inhibited by progesterone, verapamil, vinblastine, and daunomycin, indicating that the transports are due to a multidrug efflux pump (P-glycoprotein) in the canalicular membrane. Interestingly, the transport by the multidrug efflux pump was inhibited by immunosup-pressants FK506 (tacrolimus) and cyclosporine, their half-maximal inhibitory concentrations in the cell suspension being 10 μM and 0.6 μM, respectively. In contrast, the reported immunosuppressive potency of FK506 is 10− to 100-fold that of cyclosporine. Inhibition by immunosuppressants of the multidrug efflux pump, which is a transporter of cytotoxic and other drugs and present in normal human tissues—such as kidney, liver, the blood-brain barrier, and colon—may, at least partly, explain side effects caused by cyclosporine in these tissues of transplant recipients. FK506 at its clinical concentrations may not inhibit the multidrug efflux pump.