The aim of this study was to measure the activity of phase I and II key enzymes in the biotransformation of xenobiotics and their inducibility by phenobarbital (2 mM) in two currently used in vitro models, namely adult rat hepatocytes, conventionally cultured or co-cultured with rat epithelial cells derived from primitive biliary duct cells. For phase I, the cytochrome P450 content and the enzymic activities of 7-ethoxycoumarin O-deethylase and aldrin epoxidase have been determined, for phase II glutathione S-transferase activity was measured. In conventional cultures, all phase I parameters investigated declined continuously as a function of culture time. Two mM phenobarbital had inducing effects on 7-ethoxycoumarin O-deethylase and glutathione S-transferases but not on aldrin epoxidase. In co-cultures, after an initial decrease, a steady state situation developed for all the parameters measured, lasting for at least 10 days. The cytochrome P450 content, the 7-ethoxycoumarin O-deethylase, aldrin epoxidase and glutathione S-transferase activities were maintained from 3 to 4 days on at 25, 100, 15 and 50%, respectively, of their corresponding value obtained for freshly isolated hepatocytes. After phenobarbital treatment, the parameters mentioned were significantly increased with the exception of the aldrin epoxidase activity of which the inducibility was nearly completely lost.