Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein 1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of different culture conditions (suspension, conventional culture and sandwich culture) on the uptake of [(3)H]-taurocholate+/-probenecid and the efflux of [(3)H]-vinblastine+/-ketoconazole; mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was assessed by immunocytochemistry. The uptake of [(3)H]-taurocholate was higher in cryopreserved rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A significant time dependent decline in the uptake of [(3)H]-taurocholate was noticed from day 2 to day 4 in conventional and sandwich cultures. [(3)H]-taurocholate uptake was significantly reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspension was similar to that of liver, whereas much lower levels were detected in the cultures; this was in accordance with [(3)H]-taurocholate uptake results. Mdr1 activity was assessed by accumulation of the Mdr1 selective substrate, [(3)H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity.