Gonadotropins (Gn) and testosterone are important regulators of spermatogenesis, even though Gn receptors and the androgen receptor (AR) are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and CYP19 aromatase. This has further support by findings of ER expression in the testis including that of ER in spermatogonia. 5 Androstane-3, 17-diol (3Adiol), a metabolite of testosterone produced via the intermediate potent androgen 5-dihydrotestosterone (DHT), has been reported to selectively bind ER rather than ER, but not AR. Here, we have characterized the influence of 17-estradiol (E), the major physiological estrogen, 3Adiol and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3Adiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. BrdU labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E and the labeled cells were identified as spermatogonia. Moreover, high levels of 3Adiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to hCG. We suggest that 3Adiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ER–dependent pathway.