The uptake properties of taurocholate (TC) and estradiol 17beta-D-glucuronide (E(2)17betaG) were examined in freshly isolated and cryopreserved human hepatocytes to discover if active transport is retained in cryopreserved human hepatocytes. Firstly, the uptake of TC and E(2)17betaG was measured before and after cryopreservation. The uptake of TC was found to be Na(+)-dependent both in fresh and cryopreserved hepatocytes. The uptake activity in cryopreserved hepatocytes was found to range from 10 to 200% of that observed in freshly isolated cells. A kinetic analysis was performed to evaluate the transport activity of TC and E(2)17betaG and revealed that the Michaelis constant (K(m)) for these compounds in cryopreserved human hepatocytes was 2-8 and 3-18 microM, respectively. This was within the range of K(m) values previously found in human Na(+)-taurocholate cotransporting polypeptides (NTCP) and organic anion transporting polypeptides (OATP) 2 and 8, respectively. The kinetic analyses also showed that the species difference between human and rat hepatocytes was more marked for the maximal uptake rate (V(max)) (>22 and >22 times higher for TC and E(2)17betaG in rats than in humans, respectively) than that for K(m) (2-12 and 0.7-4 times higher, respectively), compared with earlier data we obtained in primary cultured rat hepatocytes. Hence, we conclude that cryopreserved human hepatocytes, at least in part, retain their transporter functions and, therefore, can be a useful experimental system for examining the mechanism of the hepatic uptake of drugs.