Phenobarbital (PB) induction of the CYP2B subfamily was studied in the livers and spleens of male and female rats. Animals were treated with either PB (10 mg/kg) or vehicle for 4 consecutive days. A reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative Northern blotting, Western blotting, and a radioenzymatic assay were used to observe differential levels of CYP2B1 and CYP2B2 mRNAs, proteins, and catalytic activities. CYP2B2 expression was limited to the livers of PB-treated male and female rats and was not detected in spleen. Low constitutive levels of CYP2B1 mRNA were markedly induced approximately 7- to 17-fold in the livers of PB-treated male and female rats, respectively. However, using the same standard oligonucleotide probe for CYP2B1 mRNA, we observed considerably greater constitutive concentrations of the transcript in spleen than in liver. Putative splenic CYP2B1 mRNA was significantly elevated by the PB treatment, although not as profoundly as the hepatic response. In contrast, only the livers of the barbiturate-treated rats expressed CYP2B1 proteins or specific catalytic activity (androstenedione 16beta-hydroxylase). Protein and catalytic activities of the isoforms were undetectable in spleen of either male or female vehicle- and PB-treated rats. In agreement, RT-PCR was unable to demonstrate the expression of splenic CYP2B1 mRNAs. Investigating the possibility that the Northern probe for CYP2B1 was identifying a similar sequence isoform, we performed RT-PCR using primers for CYP2B12 and CYP2B15. Since neither of these isoforms was expressed in spleen, we conclude that the spurious results using the Northern probe for CYP2B1 mRNA were due to the presence of a cross-reacting, PB-responsive transcript not currently identifiable in existing databases.