Ethane dimethanesulphonate (EDS) is well-recognized as a Leydig cell toxicant. Although this compound has been studied extensively to date, certain toxicological criteria have not been met. For instance, the dose-responsiveness of Leydig cells to EDS, both in vitro and in vivo, is not well established. In addition, the data regarding the cellular site of action of EDS during Leydig cell toxicity and the status of Leydig cell viability during the affected period remains controversial. The study used both highly purified (98 %) and interstitial (14 %) Leydig cell preparations to determine the in vitro EC50 (370 micro M) and in vivio (sub 50) (60 mg/kg) for hCG-stimulated testosterone (T) production, respectively. Leydig cells were recovered in approximately equal number following all in vivo and in vitro EDS exposures. Test results indicate that when Leydig cells are exposed to EDS either in vitro or in vivo, the biosynthesis of T is compromised between the production of cyclic adenosine monophosphate (CAMP) activation of protein kinase and the cholesterol side chain cleavage enzyme.