Cultures of minced, whole-ovary (whole-ovary culture) were used to determine if three selected chemicals altered steroidogenic profiles. First, phenolsulfonthalein (PST), when used in culture medium, was tested for its influence on in vitro steroidogenesis. Next, aminoglutethimide (AGTP; 0 or 150 mg/kg once) and di(2-ethylhexyl)phthalate (DEHP; 0 or 1500 mg/kg/day for 10 days) were administered in vivo to young adult cycling rats, and the ovaries and adrenals were removed and cultured for 1 h. Ovarian steroidogenic profiles of progesterone (P), testosterone (T), and estradiol (E) release into the medium were measured using radioimmunoassay techniques. PST in medium significantly altered. Therefore, PST was excluded in the later studies. DEHP altered steroid profiles so that proestrus appeared to be delayed. AGTP decreased P and E production significantly, and T production was increased slightly in proestrus ovaries. These AGTP alterations in T and E resulted in a highly significant increase in the T/E ratio. Adrenals from the DEHP and AGTP experiments were also cultured for 1 h, and P was assayed in the medium. AGTP, but not DEHP, significantly increased the production of P in adrenals. Whole-ovary culture is recommended as an in vitro test for chemicals suspected of interfering with steroidogenesis in vivo. The test model should be placed strategically between in vivo studies of reproductive toxicity and complex in vitro mechanistic studies.