Radioimmunological determination of serum LH, FSH, and estradiol concentrations in prepubertal female rats demonstrates the temporal coincidence of increased serum levels of these hormones between days 9 and 21. Serum FSH and estradiol levels are continuously high during that time, whereas interindividual fluctuations in LH levels were enormous. No high LH, FSH, and estradiol levels were observed between day 21 and puberty, during which time serum prolactin and progesterone gradually increased. Serum testosterone in the female immature rats stayed uniformly low. It is suggested that increased serum estradiol levels in the presence of low prolactin levels (between day 10 and 20) act in a positive feedback fashion on the CNS-pituitary axis. The resulting increased gonadotropin levels are later (between day 20 and puberty) decreased by an inhibitory action of prolactin and/or progesterone on pituitary gonadotropin release. In male rats serum FSH and prolactin, which were low during the first 3 weeks, increased later to reach high levels during puberty. Serum LH was slightly elevated during the 2nd and 3rd week of life at which time serum progesterone also increased to reach the highest levels in the prepubertal period. Serum testosterone was higher in male than in female rats for the first 3 weeks of life; the difference between both sexes was significant but not striking. Between day 21 and the prepubertal period the testosterone levels were relatively low, but they increased again during puberty. Sex differences in androgen levels (measured with a less specific antibody) were more pronounced whereas estradiol levels in males showed the same pattern between birth and puberty as in the female littermates. These results suggest that not only testosterone but also other, not yet identified, androgens may be involved in the masculinzation of the brain.