The maintenance effect of hexobarbital on drug-metabolizing enzyme activities were investigated in primary cultures of adult rat hepatocytes. Hexobarbital (lmM) was treated to culture media directly at the time of initial plating. The activities of drug-metabolizing enzymes, such as 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase, aminopyrine N-demethylase, and DMN demethylase were measured 24hr after culture. Hexobarbital increased the activities of drug-metabolizing enzymes in hormone-supplemented AB medium compared with WOBA-M medium. To elucidate these differences, the activities of 7-ethoxycoumarin O-deethylase of primary culture different culture conditions were examined. Hydrocortisone in AB medium significantly affected the maintenance effect of hexobarbital. The increase of drug-metabolizing enzyme activity by hexobarbital was proportional to the concentration of hydrocortisone supplemented in culture medium.