Polychlorinated biphenyls (PCBs) are environmental contaminants that in humans and animals disturb normal endocrine functions including gonadal functions. The present studies were aimed at determining the direct effects of PCB on Leydig cell testosterone production and antioxidant system in vitro. Adult Leydig cells were purified by Percoll gradient centrifugation method and the purity of Leydig cells was also determined by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining method. Purified Leydig cells were exposed to different concentrations (10(-10) to 10(-7) M) of PCB (Aroclor 1254) for 6 and 12 h under basal and LH-stimulated conditions. After incubation, the cultured media were collected and used for the assay of testosterone. The treated cells were used for quantification of cell surface LH receptors and activity of steroidogenic enzymes such as cytochrome P450 side chain cleavage enzyme (P450scc), 3beta-HSD and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In addition, Leydig cellular enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), gamma-glutamyl transpeptidase (gamma-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. The results indicated that Aroclor 1254 (10(-8) and 10(-7) M) treatments significantly inhibit basal and LH-stimulated testosterone production. In addition to this, the activity of steroidogenic enzymes, enzymatic and non-enzymatic antioxidants were significantly diminished in a dose- and time-dependent manner. Moreover, the LPO and ROS were elevated in a dose- and time-dependent manner under basal and LH-stimulated conditions. These findings suggest that PCBs can act directly on Leydig cells to inhibit testosterone biosynthesis by reducing steroidogenic enzymes, enzymatic and non-enzymatic antioxidants.