The induction of steroid 11 beta-hydroxylase and 17 alpha-hydroxylase was studied in bovine adrenocortical cell cultures in serum-free medium. In the absence of insulin-like growth factor (IGF)-I or insulin, cholera toxin failed to increase 11 beta-hydroxylase enzyme activity or messenger RNA (mRNA) levels; cholera toxin increased 11 beta-hydroxylase activity and mRNA only in the presence of 10 nM IGF-I or of higher concentrations of insulin. 17 alpha-Hydroxylase enzyme activity and mRNA, in contrast, were increased maximally by cholera toxin in the absence of insulin or IGF. We also compared the induction of 11 beta-hydroxylase and 17 alpha-hydroxylase by intracellular second messengers. When cultures were incubated with cholera toxin, cAMP analogs, forskolin, ACTH, or prostaglandin E1 in defined medium with insulin, all agents increased the mRNA levels for 11 beta-hydroxylase and 17 alpha-hydroxylase. 11 beta-Hydroxylase enzyme activity was detectable in control (insulin only) cultures and was increased to varying extents by the different agents. 17 alpha-Hydroxylase enzyme activity was undetectable in control cultures and was increased more than 50-fold by all agents. We compared the sensitivity of induction of 11 beta-hydroxylase and 17 alpha-hydroxylase enzyme activities by cAMP using serial dilutions of an equimolar mixture of N6-monobutyryl cAMP and 8-bromo cAMP. For both enzymes, the response curve was biphasic, with a maximal response in the range of 20 to 100 microM each analog, but the decline in response at higher cAMP concentrations was much more marked for 11 beta-hydroxylase than for 17 alpha-hydroxylase. The effects of activation of protein kinase C were studied in cultures incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) together with a cAMP analog mixture. TPA decreased cAMP-induced 11 beta-hydroxylase mRNA; TPA also decreased the induction of 17 alpha-hydroxylase mRNA, as previously reported. TPA caused a dose-dependent decrease in cAMP-induced 11 beta-hydroxylase enzyme activity. Angiotensin II at 0.1 to 10 microM also decreased induction of 11 beta-hydroxylase. Induction of 11 beta-hydroxylase and 17 alpha-hydroxylase is coordinately regulated by cAMP, protein kinase C, and IGF-I/insulin, but responses to these regulators differ in various respects between these two cytochrome P450 enzymes.