Abnormal sperm production and reduced fertility have been reported in transgenic male mice lacking the alpha-subtype of the estrogen receptor (ER)alpha or aromatase. The aim of this study was to investigate the role of estrogen in male reproductive function, by determining the effect of estradiol on testicular function in hypogonadal (hpg) mice congenitally lacking gonadotropin; and thus, sex steroid production. hpg mice were treated, at 2-3 months of age, with slow-release estradiol implants, which achieved circulating estradiol concentrations of approximately 40 pg/ml. Treatment for 35 days reliably induced a 4- to 6-fold increase in testicular weight, compared with the vestigial testes in the untreated or cholesterol-treated controls. The degree of testicular growth after 35 days was similar to that in hpg mice receiving an intrahypothalamic graft of preoptic area tissue taken from neonatal mice on the day of birth, a procedure known to induce testicular development in hpg mice by activation of the pituitary gland. Histological analysis revealed that the testes contained elongated spermatids after 35 days of estradiol treatment, whereas germ cell development never progressed beyond the pachytene stage in control hpg mice. Treatment for 70 days induced full qualitatively normal spermatogenesis in hpg mice. Testis weight increased 5-fold, reflecting a 5-fold increase in total seminiferous tubule volume and a 4- to 5-fold increase in the total volume of the seminiferous epithelium. In all experiments, spermatogenesis proceeded in the absence of measurable androgen concentrations, but circulating FSH concentrations were slightly (but significantly) elevated, relative to cholesterol-treated control hpg mice. This stimulatory action of estradiol on FSH secretion was unexpected, particularly because identical estradiol treatments significantly decreased serum FSH levels in wild-type littermates. These results indicate that estrogens may play a role in spermatogenesis, via stimulatory effects on FSH secretion. An alternative or complementary explanation, given the recent identification of estrogen receptors (ERalpha and ERbeta) and aromatase within various cell types in the testis, is that estrogens exert paracrine actions within the testis to promote spermatogenesis. The identification of effects of estradiol on testicular function provides a conceptual basis to reexamine the speculative link between increased exposure to environmental estrogens and reduced fertility in man.