A 57-kDa protein whose expression in rat adrenocortical microsomes is increased after weaning has been identified as cytochrome P4501B1 (CYP1B1). Levels of CYP1B1 protein were moderately expressed in late gestation fetuses and on postnatal day 1 (pdl), but were nearly undetectable on pd6 and pd1O. CYP1B1 expression initially increased in the late preweaning period (pd17-19) and again immediately postweaning (pd21-24). The temporal coincidence of CYP1B1 expression and weaning was not due to transition from suckling to solid food, as neonates that were prematurely weaned showed no increase in adrenal CYP1B1 compared with normally weaned littermates. The pattern of CYP1B1 expression paralleled changes in microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), a marker of CYP1B1 activity. Twice daily injections of ACTH to rat pups (pd3-10) failed to significantly increase the expression of CYP1B1 in pd 10 adrenals, although the injections weakly stimulated steroidogenesis. Adrenocortical cells from pd17 neonates and adult cells, when cultured for 3 days, responded similarly to ACTH induction, although neonates showed more than 4-fold less basal activity. It is concluded that rat adrenal CYP1B1 may be developmentally suppressed, and its expression is independent of diet or the presence of a dam. This suppression is retained in cell culture, but is not due to deficient ACTH signaling. These results may explain the reported resistance of neonatal rat adrenals to the toxic effects of polycyclic aromatic hydrocarbons, which are metabolized by CYP1B1 into mutagenic by-products.