Methoxychlor (MC) was developed as a replacement for the banned pesticide DDT. After in vivo administration, it is metabolized in the liver to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been shown to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen and androgen receptors, respectively. Although in vitro studies using cultured rat Leydig cells have reported that HPTE inhibits both basal and hCG-stimulated testosterone formation, the response of circulating testosterone levels to in vivo MC has been more variable. Therefore, the current studies evaluated whether the daily in vivo administration of MC (0, 5, 40 and 200 mg/kg body weight) for a short duration (days 54-60 of age) by gavage altered serum testosterone levels and ex vivo Leydig cell testosterone formation in young adult male rats. These results demonstrate that both fluid-retained and fluid-expressed seminal vesicle weights declined to 44 and 60% of control, respectively, in the 200 mg/kg MC-exposed animals. Similarly, serum testosterone and dehydroepiandrosterone levels declined to 41 and 45% of control, respectively, in the 200 mg/kg MC-exposed animals; however, serum LH and FSH levels were unaffected. Ex vivo Leydig cell basal testosterone formation over 4h declined to 49% of control in animals exposed to 200 mg/kg MC, and ex vivo Leydig cell P450 cholesterol side-chain cleavage activity declined to 79 and 50% of control in animals exposed to 40 and 200 mg/kg of MC, respectively, supporting previous in vitro studies which demonstrated the sensitivity of this step to MC.