The adrenal gland is a vulnerable target for toxic insult. Disruption of adrenal steroidogenesis and hormone secretion may cause serious effects on human health. A human in vitro model is needed to predict effects, and elucidate mechanisms of endocrine disruption and adrenal toxicity. The human adrenocortical cell line H295R has been used to screen for effects on sex hormones. Here, we have analyzed the effect of 30 potential endocrine disrupting chemicals on the secretion of cortisol and aldosterone from the H295R cells, using specific ELISA assays. The effect of chemicals was analyzed for basal and forskolin- or angiotensin II-stimulated hormone secretion. The chemicals were tested at the highest concentration where they displayed no evident unspecific cytotoxicity. Quantitative and qualitative differences in effects on hormone secretion were demonstrated for the various chemicals. A subset of the chemicals displayed different effects on cortisol and aldosterone secretion, and in some cases the effects were different between basal and stimulated hormone secretion. Aminoglutethimide, prochloraz, ketoconazole, 6-hydroxyflavone, imazalil and etomidate had the most marked inhibitory effects on cortisol (with or without forskolin) and ketoconazole, 6-hydroxyflavone, imazalil and etomidate had the most marked effects on aldosterone (with or without angiotensin II). The results are discussed in terms of known effects, structural similarity and possible mechanisms. We have shown that adrenal steroidogenesis is a vulnerable target for toxic insult and that the H295R assay is a useful in vitro model for screening purposes.