Cytochrome P450-mediated 4-hydroxylation is an important pathway in the termination of the biological action of retinoids. Several purified mammalian hepatic P450s have been shown to catalyze the 4-hydroxylation of all-trans-retinoic acid (retinoic acid) in reconstituted enzyme systems, but the nature of the activity in untreated rat liver microsomes has not been defined. In the present study, microsomal retinoic acid 4-hydroxylation was characterized in untreated liver from rats of both sexes and after a series of induction treatments. Thus, dexamethasone and phenobarbital, but not beta-naphthoflavone or dimethyl sulfoxide, increased the activity in male and female rats. An immunoglobulin G (IgG) fraction raised against P450 3A1 decreased the rate of retinoic acid 4-hydroxylation in untreated rat hepatic microsomes, but IgG directed against the P450s 2C11, 2B1, and 2C6 were noninhibitory. In vivo administration of triacetyloleandomycin, which has been shown to form a metabolite intermediate complex with P450 3A, followed by potassium ferricyanide oxidation in vitro, reactivated retinoic acid 4-hydroxylation and androst-4-ene-3,17-dione 6 beta-hydroxylation similarly (to 154 and 152% of the respective activities in the absence of potassium ferricyanide). Finally, exogenous retinoic acid (60 mg/kg ip for three days) markedly increased the rate of retinoic acid 4-hydroxylation in hepatic microsomes from male and female rats (3.8- and 3.7-fold, respectively). This occurred without comparable increases in other P450 activities. Despite these findings, the age- and sex-related profiles of microsomal retinoic acid 4-hydroxylation were clearly different from those measured for other P450 activities. Thus, on the basis of xenobiotic pretreatment, developmental, and immunochemical studies, the enzyme(s) involved in constitutive retinoic acid 4-hydroxylation appears distinct from a number of well-described P450s in untreated and induced rat liver. The data are consistent with the partial involvement of P450(s) from the 3A subfamily in retinoic acid 4-hydroxylation, but it seems clear that P450s 3A1 and 3A2 do not participate in this activity.