In vitro whole-ovary culture in rats was used to characterize ovarian steroidogenesis and to evaluate changes produced by in vivo exposure to bis(2-diethylhexyl) phthalate (DEHP). Steroidogenic profiles (progesterone (P4), estradiol (E2), and testosterone (T)) from minced ovary cultures were obtained in untreated immature and mature rats, and from mature rats treated with DEHP. A one-hour incubation without human chorionic gonadotropin (hCG) was used to produce a basal steroidogenic profile. Three one-hour incubations with hCG were used to produce a stimulated profile. Using a multivariate statistical analysis, a combination of basal and stimulated ovarian steroid profiles correctly identified the stage of the cycle all untreated rats. Alone, basal or stimulated ovarian steroid profiles correctly identified the stage of the cycle in more than 90% of the rats. The statistical analysis using a combination of variables (multivariate) indicated that DEHP treated rats were significantly different (P < 0.001) from sham treated rats. In fact, the alteration caused by DEHP in the in vitro ovarian steroidogenic profile was most apparent in rats on vaginal diestrus and estrus. In DEHP-treated rats in diestrus, ovarian steroidogenesis appeared to shift to the production of more T and E2 than in untreated rats in diestrus.