In this study the NCI-H295R human adrenocortical carcinoma cell line was used as an in vitro biological model to study the effect of mercury (HgCl(2)) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 μM of HgCl(2) and compared to control. Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity. Quantification of testosterone and progesterone directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Concentration-dependent depression in testosterone production was detected particularly for higher concentration of Hg(2+). Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 μM) of Hg(2+) this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 μM and higher concentration of Hg(2+). However, at 25 μM Hg(2+) exposure the cell viability remained relatively high (> 80%). Results of the study indicate dose-dependent decreases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl(2) exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.