The separation of the prepuce from the glans penis, termed preputial separation, has been shown to be androgen dependent and to occur around the time of puberty in rats. In the present studies the mean ages of preputial separation in four experiments were 39, 42, 44 and 45 days of age, just preceding the appearance of mature sperm in the caput epididymis. Preputial separation occurred prior to the significant increase in circulating androgen levels by Day 51 as measured by radioimmunoassay. However, castration as late as Day 35 blocked preputial separation demonstrating that separation of the prepuce was dependent on the continued presence of a low level of androgen. Silastic implants of testosterone or dihydrotestosterone, but not estradiol, reverse the effects of castration. Handling neonatal males every day prior to weaning significantly delayed preputial separation and this delay was correlated with significantly lower circulating levels of androgen and LH at 45 days of age. These data show that preputial separation, an easily determined external sign of sexual development in male rats may be used as an index of change in peripubertal androgen secretion.