Hint2 belongs to the superfamily of histidine triad (HIT) hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immuno blot analysis of subcellular fractions. The protein appeared associated to mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by AngII or K(+), while protein silencing with specific siRNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by AngII was also reduced upon Hint2 down regulation with siRNA, but not affected after its overexpression, suggesting that under basal conditions Hint2 is optimally expressed, and not rate-limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca(2+)-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analogue of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favourable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.