An earlier study from this laboratory showed that the hepatic murine cytochrome P450 (P450) system was depressed by interferon in vivo but induced in cultured primary hepatocytes. The current investigation attempted to resolve this contradiction. The P450 content of the cells used in the earlier study fell precipitously during the first 24 hr of culture and remained at the same low level throughout another 48 hr of incubation. This failure to maintain the P450 level suggested that the cells may not have been sufficiently viable to support the mechanisms involved in the depressant activity of interferon. Accordingly, a chemically defined medium containing hydrocortisone was devised which supported an acceptable level and function of the P450 system throughout a 72 hr incubation period. Functionality of the P450 system was evaluated by measuring aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities. When this steroid supplemented medium was used, interferon depressed both activities by about 25%; however, neither activity was affected significantly by poly IC. On the other hand, benzo[a]pyrene hydroxylase activity was depressed by both poly IC and interferon in hepatocytes induced with dexamethasone or with dexamethasone plus 3-methylcholanthrene. These studies emphasize the necessity of maintaining an acceptable level of homeostasis in cultured hepatocytes if one is to derive meaningful interpretations of certain biological events.