Although the persistent pesticide DDT has been banned or restricted since the 1970s, DDT and its metabolites may still pose a hazard to wildlife and humans. There is a growing concern regarding endocrine disruptors and their potential role in negative trends in male reproductive health. The aim of this study was to investigate the effects of two DDT metabolites, 3-MeSO2-DDE and o,p’-DDD, and the synthetic DDE analogue 3,3’-(bis)MeSO2-DDE on mammalian male reproductive system by evaluate the effect on testicular steroidogenesis in LH-stimulated neonatal porcine Leydig cells in vitro. The test compounds are known to disrupt steroidogenesis and cause toxicity in the adrenal cortex of several species, and o,p’-DDD is used in the treatment of adrenocortical cancer in humans. 3-MeSO2-DDE has been suggested as an alternative drug and 3,3’-(bis)MeSO2-DDE might reveal any structure-specific effects regarding methylsulfonyl moiety on the steroidogenesis. Testicles were obtained from routine castrations of neonatal piglets and Leydig cells were isolated and purified using a discontinuous Percoll gradient. The primary cultures of Leydig cells were stimulated with LH and exposed to the test compounds for 48 hours in six increasing concentrations (0-20 μM). Cell viability was assessed and the production of testosterone, estradiol, progesterone and cortisol was measured from the cell medium. Gene expression analysis was performed on 16 genes involved in testicular Leydig cell steroidogenesis. The results showed that both 3-MeSO2-DDE and o,p’-DDD were cytotoxic at the highest concentration (20 μM), whereas 3,3’-(bis)MeSO2-DDE had no effect on cell viability. All three test compounds decreased LH-stimulated production of testosterone, estradiol and progesterone by the Leydig cells in a concentration-dependent manner. No detectable levels of cortisol were obtained. The gene expression analysis suggested a general suppression in mRNA levels of several genes involved in steroidogenesis induced by all three test compounds. However, the compounds reduced expression of slightly different genes. The decreased expression of some genes involved in steroidogenesis might be associated with the reduced hormone production in exposed cells. This study suggests a steroidogenic disruption in porcine Leydig cells induced by the test compounds. The suppression of Leydig cell steroidogenesis by the two DDT metabolites might be of concern regarding negative effects on male reproduction.