Testosterone is essential for quantitatively normal sperm production in the testis, normal sperm maturation in the epididymis, maintenance of the accessory sex organs, and effective sexual behavior. A variety of xenobiotics can result in a significant decrease in spermatogensis, sperm motility and fertility, libido, or simply the circulating level of testosterone. Thus, the ability to assess the steroidogenic capacity of the Leydig cell is pivotal to a complete characterization of toxicant-induced effects on reproductive function in the male. Previously, it was impossible to conduct definitive studies to identify direct toxicant-induced effects on Leydig cell function and viability since a method to provide viable, highly purified Leydig cell preparation was unavailable. Herein the authors describe such an isolation procedure as well as criteria for maintaining Leydig cells in primary culture. A primary culture of Leydig cells which maintains function over time, provides a model for those interested in addressing the more mechanistic issues in Leydig cell toxicology and permits the determination of the reversibility of toxicant-induced effects in vitro.