Testis and ovary explants have been proposed asin vitroscreens for identifying potential inhibitors of steroid biosynthesis. The goals of the current study were to optimize the conditions of the two assays, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the responses from the explant assays with anin vivomale battery currently undergoing validation using the Crl:CD BR rat in order to evaluate their utility as test systems for screening unknown compounds for possible steroid biosynthesis inhibition activity. There were two components to the testis/ovary assays:ex vivoandin vitro.Theex vivocomponent used testes/ovaries from animals dosed with the test compoundsin vivo,and thein vitrocomponent used testes/ovaries from control animals. For the testis assays, decapsulated testis explants (50 mg) were placed into glass scintillation vials, ±1.0 IU/ml hCG for 3 h in a shaking water bath (34°C). Following the incubation period, medium was removed, centrifuged, and frozen until assayed for hormone concentrations. A similar procedure was used for the ovary explant assay except that each ovary was incubated separately. The testis explants were evaluated using the following compounds: ketoconazole (KETO), a testosterone biosynthesis inhibitor; aminoglutethimide (AG) (onlyin vitro) and anastrozole (ANA), aromatase inhibitors; finasteride (FIN), a 5α-reductase inhibitor; 17β-estradiol (17β-E2), an estrogen receptor agonist; flutamide (FLUT), an androgen receptor antagonist; ICI-182,780 (ICI), an estrogen receptor antagonist; haloperidol (HALO), a D 2 receptor antagonist; and reserpine (RES), a dopamine depletor. In the ovary assay, AG (onlyin vitro), ANA, ICI, and HALO (onlyin vitro) were evaluated. Addition of fetal calf serum to the medium allowed measurement of estradiol (E2) in the testis assay, but production was not inhibited by ANA or AG. In the ovary explant assay, only AG was identified as inhibiting E2 productionin vitro.Hence, both the testis and ovary explant assays appear to have limited utility for detecting aromatase inhibitors. Screening of these nine diverse endocrine-active compounds resulted in all of them being identified as altering the endocrine system when assessed byex vivoandin vitrotestis explants. Using only thein vitroassessment with the criteria of steroid biosynthesis inhibition, four of nine compounds were correctly identified in the testis explant assay (17β-E2, KETO, FLUT, and HALO). The predictability of both thein vitroandex vivoovary assay was 50%, suggesting a 50% false positive or negative rate with unknown compounds. However, of the seven compounds assessed to date (17β-E2, ICI, ANA, KETO, FLUT, HALO, and RES), all were correctly identified using anin vivomale battery, which also has the capability to detect other endocrine activities. Therefore, the testis and ovary explant assay would not be necessary if one were using anin vivomale battery, since this screen would identify steroid biosynthesis inhibitors and would also identify several other endocrine activities. Because of the difficulties in assessing cytotoxicity and the high false positive/negative rates, the ovary and testis explant assays are not useful as routine screening procedures for detecting steroid biosynthesis inhibitors; however, they may have utility in confirmingin vivofindings.