Cytochrome P450 2B1, encoded by CYP2B1, and cytochrome P450 2B2, encoded by CYP2B2, are inducible in rat liver by phenobarbital (PB). We have used cultured adult rat hepatocytes to study molecular mechanisms regulating CYP2B1/CYP2B2 transcription. Northern blot analysis demonstrated that the endogenous CYP2B1/CYP2B2 genes were inducible by PB in such cultures. A PB-responsive element (PBRE) conferring PB inducibility on a reporter gene was identified in the CYP2B2 5'-flanking region. The PBRE was localized to a 163-bp Sau3AI fragment situated between 2155 and 2318 bp upstream from the CYP2B2 transcription start point (tsp). The PBRE also conferred PB responsiveness on an enhancerless heterologous promoter and was active in both orientations both upstream and downstream from the heterologous promoter; hence, it has the properties of a transcriptional enhancer. Gel-retardation assays showed that nuclear extracts of liver cells of untreated and PB-treated rats contained sequence-specific DNA-binding factors that interact with a PBRE-containing DNA fragment. These results may open the way to identifying one or more transcription factors mediating induction of CYP2B2 and CYP2B1 in rat liver.