Objective: To determine changes in adrenal androgen (AA) production, and transcription of dehydroepiandrosterone (DHEA) sulfotransferase (SULT2A1) in the NCI-H295R human adrenocortical cell line in response to insulin and testosterone, an environment mimicking the polycystic ovary syndrome state. Design: In vitro experiment using NCI-H295R adrenocortical cell lines. Setting: Academic medical center. Patient(s): NCI-H295R human adrenocortical cell line. Intervention(s): The transcriptional activity of SULT2A1 and adrenal steroid production was quantified after exposure to various treatments (e.g., forskolin, insulin, testosterone, and combinations thereof). Main Outcome Measure(s): Quantification of mRNA for DHEA sulfotransferase (SULT2A1) by real-time reverse transcription-polymerase chain reaction and measurement of steroid production by radioimmunoassay. Result(s): Testosterone decreased DHEAS and cortisol, and increased DHEA secretion by H295R cells; the inhibitory effects of testosterone on DHEAS and cortisol production were augmented by insulin. There was a trend toward an increase in the transcription of SULT2A1 by insulin and testosterone. Conclusion(s): Testosterone and insulin appear to be modulators of adrenal androgen production in this human adrenocortical cell model. These results suggest that testosterone may augment DHEA secretion in the human adrenal, although they do not support the role of this sex steroid or insulin on the elevated DHEAS levels frequently observed in polycystic ovary syndrome. © 2009 American Society for Reproductive Medicine.