Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.