In order to study the sequence of hormonal changes that accompany the onset of puberty in the female rat, immature animals were sacrificed by decapitation between days 32 and 38, and plasma titers of gonadotropins, prolactin, and LHRH, and the hypothalamic content of LHRH were determined by specific radoimmunoassays (RIA). Animals were decapitated at 10:00 and 16:00 h throughout the pubertal period, the uterine weight was recorded, and the ovaries were inspected for signs of ovulation. Animals with the vagina closed were grouped according to the condition of the uterus as anestrus (A), early proestrus (EP) and late proetrus (LP), the uterus being unstimulated, dilated with some fluid, or ballooned, respectively. Vaginal opening was usually associated with ovulation and in most cases occurred at the end of the late proestrous phase. Animals were studied up to 3 days after vaginal opening and were grouped according to vaginal cytology. Uterine weight, taken as an index of estrogen secretion, was low during A, increased during EP, and reached a peak at LP, declining thereafter. Plasma LH and FSH were low from A until the afternoon of LP. At this time, uterine weight was maximal and both gonadotropins increased dramatically. The following morning (estrus), LH but not FSH, had returned to basal values. FSH returned to basal levels on the afternoon of estrus. Plasma prolactin was low in the morning during the entire period, but showed peaks on the afternoon, which reached a maximum at LP and declined thereafter following the pattern of changes in uterine weight. Plasma LHRH was uniformly low throughout the entire pubertal period, whereas hypothalamic LHRH content declined on the morning of estrus (day of vaginal opening). We suggest that the onset of puberty in the female rat is brought about by a gradual increase in estrogen secretion which, acting at the CNS-pituitary level, triggers a preovulatory proestrus-like surge of gonadotropins and prolactin.