Hypothesis: Stress is thought to disrupt the balance between prooxidants and antioxidants by generating reactive oxygen species damaging the testicular tissue and steroidogenesis. Objective: The in vitro model of isolated and cultured Leydig cells of male Sprague Dawley rats was designed to investigate the effects of antioxidants, ascorbic acid and alpha tocopheral separately and combined with stress, hormones [corticosterone and nor epinephrine] at Leydig cell level. Methods: Four rats were taken per experiment, decapitated and orchitectony was done. After decapsulation and enzymatic dispersion, density gradient Percoll centrifugation method was used and through various steps, we obtained purified Leydig cells with 85,000 cells per culture tube. Purified Leydig cells were incubated with corticosterone [100 nmol/tube], nor epinephrine [10 umol/well], antioxidants ascorbic acid [100 umol/ml] and alpha tocopherol [10 ug/ml] in the presence of LH [50 ul/week]. The testosterone production and extent of lipid peroxidation and superoxide dismutase activity were evaluated by enzyme immunoassay. Data were analyzed by SPSS version 17 and independent sample t test were applied to find difference among control and treated wells. Results: The data revealed that corticosterone decreased the Leydig cell steroidogenesis through non genomic rapid pathways because we used 3 hours short term cell culture model and genomic pathways need longer duration than this. Whereas, nor epinephrine increased testosterone synthesis at Leydig cell level beyond pregnenolone which we observed after addition of pregnenolone to the culture tubes. Combination of corticosterone and nor epinephrine resulted in decreased [p value< 0.05] testosterone production. The anti-oxidants like ascorbic acid and alpha tocopherol when superadded alone, revealed favourable preventive effects at Leydig cell level and blunted the fall in testosterone and rise in malondialdehyde levels. Combination of antioxidants, ascorbic acid and alpha tocopherol revealed synergistic effect in reducing oxidative stress (p value< 0.001) and prevented stress hormones induced derangements in testicular steroidogenesis. Conclusion: The antioxidants, ascorbic acid and alpha tocopherol decrease the stress induced lipid peroxidation and prevent the decline in testicular steroidogenesis presumably by stabilizing the membrane of the Leydig cells.