Intracellular accumulation at 37 degrees C of 50 microM [14C]taurocholic acid by hepatocytes of pig and rat, cultured for 24 hours, and by human hepatocytes, cultured for 12 hours, reached equilibrium after an incubation time of 1 to 2 hours. Maximum capacity to accumulate taurocholate intracellularly was assessed in 3-hour incubations with increasing extracellular taurocholate concentrations. Accumulation capacity of pig and rat hepatocytes was saturated at 100 microM, while uptake by human hepatocytes slightly increased even further above this concentration. At extracellular concentrations of 100 to 500 microM, hepatocytes of these three species concentrated taurocholic acid intracellularly to between 13 and 17 nmol per mg cell protein, corresponding to an intracellular concentration which was 10-70 times higher than the added extracellular concentration. With proceeding culture age, accumulation capacity of rat and human hepatocytes declined steeply (-80% and -60%, respectively between the first and second culture day). In contrast, in cultured pig hepatocytes, this capacity was only 40% lower on the third day compared to the first day of culture. It is concluded that in cultured pig hepatocytes, the capacity to accumulate bile acids is retained for a longer time than in cultured rat and human hepatocytes.