The presence of estrogen receptor beta and aromatase in the germ cell has highlighted the physiological role of the traditionally female hormone, estrogen, in spermatogenesis. Estrogen receptor alpha knockouts and aromatase knockouts have further accentuated the role of estrogen in germ cell maturation. To delineate the direct action of estrogen in the seminiferous epithelium, we studied the effects of high intratesticular estradiol. The study was based on the fact that administration of exogenous estradiol suppresses the hypothalamus pituitary gonadal axis (HPG) with a dose-dependant concomitant increase in intratesticular estrogen levels. Three doses of 17-beta estradiol, namely 20, 100 and 200 microg/kg/day were administered subcutaneously to different batches of adult male rats for 10 days. The effect of the three doses on serum hormonal profile, intratesticular testosterone (T) and estradiol (E) levels were studied. Twenty micrograms per kilograms per day of 17-beta estradiol affected the hypothalamus-pituitary axis, reducing serum gonadotropins and intratesticular testosterone; however, 100 microg/kg/day of 17-beta estradiol decreased serum FSH and intratesticular testosterone, increased intratesticular estradiol, but had no effect on serum LH. Interestingly, 200 microg/kg/day of 17-beta estradiol decreased serum and intratesticular T without any effect on serum gonadotropins. This could be attributed to the positive feedback effect of estrogens on gonadotropins. In the testis, morphologically two visible effects were seen, namely 'spermiation failure' in all three doses attributed to the suppression of T and FSH and a 'maintenance effect' in the 100 microg/kg/day attributed to E and/or 10% of available intratesticular T. The direct effect of an increase in intratesticular estradiol levels was observed in terms of a decrease in apoptosis in germ cell. The study, therefore, suggests that 100 microg/kg/day of 17-beta estradiol could be used to study the effects of high intratesticular estradiol with a concomitant decrease in intratesticular T and serum FSH levels on spermatogenesis.