The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the development of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carcinoma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR. Treatment with forskolin increased cortisol secretion and stimulated transcription of all the steroidogenic genes except SULT2A1. The transcript profile from H295R cells in the presence and absence of forskolin was compared with the transcript profile from human adrenal glands. The gene expression pattern observed in the forskolin-treated H295R cells was more similar to that in the human adrenal gland, than the expression pattern in untreated cells. To examine H295R cells as a possible in vitro system for the assessment of adrenal disruption using molecular endpoints, the insecticide lindane (gamma-hexachlorocyclohexane) was used. In vivo, lindane has been shown to inhibit testicular, ovarian and adrenal steroidogenesis. It was demonstrated that lindane reduced cortisol secretion, downregulated the expression of a subset of the genes encoding steroidogenic enzymes and repressed transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene promoter. Thus the H295R cell line provides a good in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression. This in vitro test can be used for the rapid detection of adrenal endocrine disruption and as a tool for mechanistic studies.