Tumor necrosis factor- (TNF-) regulates the hypothalamo-pituitary-adrenal axis at several levels. It has been shown to modify adrenal steroidogenesis in many species, and it is supposed to act as an auto/paracrine factor. However, its significance in human adrenocortical function remains unclear. We therefore investigated the effect of TNF- on adrenal steroidogenesis, expression of the key steroidogenic genes, apoptosis, and cell viability in the human adrenocortical cell line NCI-H295R. TNF- treatment (1 nM for 48 h) decreased the basal production of cortisol, androstenedione, DHEAS and aldosterone (14, 18, 35 and 52%, respectively) and the 8-Br-cAMP induced production of cortisol, androstenedione, DHEA and DHEAS (44, 66, 58 and 48%, respectively). However, when the steroid production data were normalized by the cell number, TNF- increased the basal production of cortisol, androstenedione, DHEA, DHEAS and aldosterone (137, 121, 165, 73 and 28%, respectively), and the 8-Br-cAMP induced production of cortisol, DHEAS and aldosterone (122, 121 and 256%, respectively). This was accompanied by a parallel increase in the expression of the genes encoding for the steroidogenic acute regulatory protein, 3-hydroxysteroid dehydrogenase 2 and 17-hydroxylase/17,20-lyase (74, 200, and 50%, respectively; quantitative real-time RT-PCR analysis). TNF- increased caspase 3/7 activity (an indicator of apoptosis) and decreased cell viability dose- and time-dependently. The effect of TNF- on apoptosis was neutralized by a monoclonal TNF- antibody. These findings indicate that TNF- is a potent regulator of steroidogenesis and cell viability in adrenocortical cells. TNF- may have physiological and/or pathophysiological significance as an endocrine and/or paracrine/autocrine regulator of adrenocortical function.