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To identify and prioritize chemicals that may alter steroidogenesis, an in vitro screening assay based on measuring alterations in hormone production was developed using the H295R human adrenocortical carcinoma cell line. Previous studies indicated that this cell line was useful to screen for effects on gene expression of steroidogenic enzymes. This study extended that work to measure the integrated response on production of testosterone (T), estradiol (E2), and progesterone/pregnenolone (P) using an ELISA. Under optimized culture and experimental conditions, the basal release of P, T and E2 into the medium was 7.0 ± 1.2 ng/ml, 1.6 ± 0.4 ng/ml, and 0.51 ± 0.13 ng/ml, respectively. Model chemicals with different modes of action on steroidogenic systems were tested. Exposure to forskolin resulted in dose-dependent increases in all three hormones with the greatest relative increase being observed for E2. This differed from cells exposed to prochloraz or ketoconazole where P concentrations increased while T and E2 concentrations decreased in a dose-dependent manner. In cells exposed to fadrozole, E2 decreased in a dose-dependent manner while T and P only decreased at the greatest dose tested. Aminoglutethimide decreased P and E2 concentrations but increased T concentrations. Vinclozolin reduced both P and T but resulted in a slight increase in E2. The alteration in the patterns of hormone production in the H295R assay was consistent with the modes of action of the chemicals and was also consistent with observed effects of these chemicals in animal models. Based on these results, the H295R in vitro system has potential for high throughput screening to not only characterize the effects of chemicals on endocrine systems but also to prioritize chemicals for additional testing.