A chemically defined medium developed for the maintenance of differentiated adult rat hepatocytes (T1) was compared with two commercially available media (Waymouth 752/1 and Leibovitz L-15) for maintenance of cytochrome P-450 metabolic activity in cultured hepatocytes. Specific metabolic activities of initially isolated cells and 72-hr control and phenobarbital-treated cultures were determined with 7-ethoxycoumarin, 7-ethoxyresorufin, and 7-pentoxyresorufin as substrates. Control and phenobarbital-treated cultures in T1 medium had a higher metabolic activity towards each of the three substrates than comparable cultures in the other media. These studies indicated that the metabolic activity and the response to phenobarbital of the major isozyme of the phenobarbital-inducible family of cytochrome P-450 were maintained in hepatocytes in T1 medium. However, there was anomalous expression and induction by phenobarbital of the major 3-methylcholanthrene-inducible isozyme, cytochrome P-450c, in cultured hepatocytes in each of the three media tested, but this response was more pronounced in T1 medium. In conclusion, the regulation of cytochrome P-450 metabolic activity in cultured hepatocytes was shown to be dependent on the composition of the culture medium.