The cause of the onset of puberty in the rat or any other mammalian species is unknown. According to one theory, puberty is initiated through switching of the brain "gonadostat." It is hypothesized here that puberty in the rat is the consequence of the appearance of free, and therefore physiologically active, estrogen in the circulation. To test this, the unbound fraction of estradiol in serum of immature female rats was measured in relation to the nuclear receptor occupancy of estradiol in the hypothalamus, preoptic area, and uterus at various times after birth. In addition, an attempt was made to alter the free fraction of estradiol by injection of the estradiol-binding protein alpha-fetoprotein (AFP) into immature female rats. The free fraction of estradiol was low (less than 1%), but began rising at about 20 days of age, and a significant increase in nuclear bound estradiol was observed in 23-day-old rats (P less than 0.001). By day 30, unbound levels attained adult values (3.99 +/- 0.15%). At this time, nuclear bound estradiol in all tissues examined fell (P less than 0.01), but by day 40, these were greatly increased in rats in estrus (P less than 0.001), being trebled in the preoptic areas and doubled in the hypothalamus. Injection of AFP into immature female rats extended the period of low free estradiol (1.22 +/- 0.08%), while in albumin-injected rats, the free fraction was 4.44 +/- 0.1%. Injection of AFP resulted in levels of nuclear-bound estradiol that were less than half those measured in nuclei from AFP-injected animals (P less than 0.001), and AFP delayed puberty. The affinity of the reaction between estradiol and nuclear receptors in brains of immature and mature rats was not significantly different; the Kd fell within the range of 0.05-0.08 nM. It is suggested that in the rat, puberty is the result of the appearance in the circulation of physiologically active estradiol after day 20.