The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.