The effect of insulin on the phenobarbital (PB)-induced gene expression of CYP2B1 and CYP2B2 (CYP2B1/2B2) in adult rat hepatocytes was investigated. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was found to strongly suppress the induction of CYP2B1/2B2 gene expression in hepatocytes cultured on EHS-gel. Although the induction by PB was not seen in monolayer hepatocytes cultured on type I collagen under standard culture conditions, the induced expression of the CYP2B1/2B2 gene was observed in monolayer hepatocytes by removing insulin from the medium. Further, we succeeded in maintaining the prolonged induction of CYP2B1/2B2 by PB in monolayer hepatocytes by using a medium containing dexamethasone but not insulin. Since the PB-induced UDP-glucuronosyltransferase gene expression was not reduced by insulin, the suppressive effect of insulin was considered to be specific to the CYP2B1/2B2 gene. These results demonstrate that insulin in media masks the PB-induced expression of the CYP2B1/2B2 gene in conventional monolayer hepatocytes and that the use of insulin-free media with primary hepatocytes provides a useful tool for investigating the molecular mechanism of CYP2B1/2B2 gene expression.