Cytochrome P4501B1 (CYP1B1), which is responsible for metabolism of 7,12-dimethylbenz[a]anthracene in the rat adrenal gland, is partially dependent on ACTH in vivo. The regulation of CYP1B1 and possible involvement in steroidogenesis have been characterized in cultured rat adrenocortical cells. Relatively high basal expression of CYP1B1 is maintained in vitro and is, therefore, independent of ACTH. CYP1B1 expression is elevated 4-fold in primary cultures of fasciculata cells after 24 h of ACTH treatment, as measured by selective 7,12-dimethylbenz[a]anthracene metabolism, immunoblot analysis, and parallel changes in the 5.2-kilobase CYP1B1 messenger RNA (mRNA). Corticosterone synthesis was stimulated about 40-fold in these cells after this ACTH treatment. Maximal stimulation of CYP1B1 protein and mRNA by ACTH has been duplicated in fasciculata cells by 8-bromo-cAMP and the adenylyl cyclase agonist, forskolin, indicating that cAMP mediates this induction. CYP1B1 is similarly stimulated by ACTH in rat adrenal glomerulosa cells, although constitutive expression of CYP1B1 is about 4-fold lower. Angiotensin II treatment of glomerulosa cells, which stimulated aldosterone synthesis 3-fold, had no effect on CYP1B1 activity or expression. Treatment of fasciculata and glomerulosa cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in small increases in CYP1B1 activity (1.8- and 2.5-fold, respectively), but much larger increases (5- and 6-fold, respectively) in CYP1B1 at the mRNA level 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect in the presence of ACTH stimulation. CYP1B1 is not coordinately expressed with steroidogenic enzymes. CYP11A1 and CYP21 mRNAs are far more responsive to ACTH, in part because of lower basal expression. CYP1B1 exhibited a transient response to ACTH that peaked (9-fold) at 6 h before declining to about 4-fold at 36 h, the time when CYP21 mRNA was maximally stimulated. The complete inactivation of CYP1B1 activity in fasciculata cells by a mechanism-based inhibitor, 1-ethynylpyrene, did not affect corticosterone production, indicating that this protein does not have a direct physiological role in the steroidogenic response.