An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.