Isolated rat hepatocytes have been advocated as a model to study aspects of mechanism of chemical-induced interference with biliary excretory function. Some technical problems do exist in studying efflux, such as the reuptake of the previously excreted substrate. Another concern is the loss of liver-specific functions in hepatocytes with continuing time in culture. It is important to address such technical aspects and to determine whether the process of efflux is compromised in primary cultures of hepatocytes. In the presence of Na+ the apparent efflux of taurocholate from hepatocytes was shown to be significantly confounded by reuptake of substrate. The unidirectional efflux was best demonstrated in buffer where choline replaced Na+. A comparison of efflux kinetics for cultured cells to those in suspension showed that both apparent affinity for transport carriers and transport capacity were greater in the former. The simple diffusion component for efflux increased with the time in culture, but affinity for transport carriers and transport capacity remained unchanged over 6 to 24 hr. However, it was not possible to determine meaningful kinetic constants after 24 hr in culture because the uptake of taurocholate was so low. Primary cultured hepatocytes may therefore be of limited value in the study of efflux of bile salts in the longer term, mainly because of the inability of cells to take up and accumulate a sufficiently high level of bile salts.