Administration of low doses of beta-naphthoflavone (beta-NF) to pregnant rats on days 7 to 14 of gestation has been associated with extensive fetal mortality. Studies were conducted to evaluate the effects of beta-NF administration on several extrahepatic cytochrome P-450-dependent enzymes responsible for steroid biosynthesis in the ovary. Pregnant rats received beta-NF (15 mg/kg) for 2-, 4- or 6-day periods before study on day 15 of gestation. At this stage of pregnancy no adverse effects on maternal weight gain, percent resorptions or fetal body weight were observed after beta-NF treatment. Cytochrome P-450 content of ovarian microsomes was not altered by beta-NF treatment. Estrogen biosynthesis in ovarian microsomes (aromatase activity), assayed as conversion of [14C]testosterone to 17 beta-estradiol, was increased nearly 2-fold in the beta-NF-treated dams. Incubation of ovarian microsomes with [14C]progesterone yielded 20 alpha-hydroxy progesterone and an unidentified compound as major products. There was no evidence of cytochrome P-450-dependent conversion of progesterone to androgens at this stage in pregnancy. In contrast to the effects of beta-NF on aromatase activity, progesterone catabolism was unaltered after administration of beta-NF for a 4-day period. The absence of a change in 20 alpha-hydroxy steroid dehydrogenase activity suggests that corpus luteum function is unaltered after beta-NF exposure. These data indicate that beta-NF administration during midgestation is associated with a selective alteration in in vitro ovarian steroidogenesis and further suggest a mechanism for beta-NF related in utero toxicity.