Today we realize that by elucidating the underlying toxicological mechanisms of toxicant action, we can greatly improve our ability to accurately assess human risk. After adequate dose-response and target organ data are gained from in vivo toxicant exposures, validated in vitro models are appropriate to study the sequelae of subcellular effects elicited by the toxicant. In many cases of spermatogenic dysfunction Sertoli and/or Leydig cell compromise is indicated. We describe herein the current in vitro models for examining Sertoli and Leydig cell function during xenobiotic exposure. These methods have already served to shed much light on the mechanism of action of ethane dimethanesulphonate, a well known Leydig cell toxicant, and the phthalates which are cytotoxic to Sertoli cells. Future research utilizing these in vitro models will undoubtedly lead to an awareness of homologous mechanisms of toxicant action between rodents and humans, knowledge which will ultimately be used to develop biomarkers for effects.