The aim of the present study was to identify the sites of the inhibitory action of TNFalpha (tumor necrosis factor alpha) on LH/hCG-stimulated testosterone formation. By using cultured porcine Leydig cells as a model, TNFalpha was shown to inhibit testosterone secretion when testicular cells were stimulated with hCG but not when incubated with 22R-hydroxycholesterol (a cholesterol substrate derivative that readily passes through cell and mitochondrial membranes). Such an observation suggested that the cytokine may affect cholesterol transport and/or availability to cytochrome P450scc in the mitochondria. Specifically, we report here that TNFalpha reduced in a dose- and time-dependent manner hCG-induced StAR (steroidogenic acute regulatory protein) levels. The maximal and half-maximal effects were obtained with 20 ng/ml (1.2 nM) and 1.6 ng/ml (0.09 nM) of TNFalpha, respectively. Maximal inhibitory effects of TNFalpha on StAR messenger RNA and protein levels were obtained after 48 h of treatment. Additionally, the presence of TNFalpha receptors P55 in terms of protein (identified through cross-linking experiments) and messenger RNA (identified through RT-PCR analysis) suggested that the effects of the cytokine are directly exerted on the testicular steroidogenic cell type.