By using serum-free co-culture of Leydig cells and Sertoli cells isolated from Rhesus Monkey(Macaca mulatta),we analyzed the effect of adrenocorticotropic hormone(ACTH),luteinizing hormone(LH),cAMP,endorphin,and naloxone on the secretion of estradiol. We showed that ACTH,LH,cAMP,and naloxone stimulated estradiol production in co-cultured system and this stimulation was enhanced by increased Leydig cells. We also showed that endorphin inhibited estradiol secretion by Leydig cells and Sertoli cells when a certain amount of Leydig cells were present. Thus,these results suggest the amount of Leydig cells may play an important role in estradiol production in Leydig-Sertoli cells co-culture.