When administered during the critical perinatal period, estrogen permanently modifies both male and female reproductive function. This study evaluated the influence of exogenous estrogen administered during this time on the hypothalamic LHRH content and on gonadotropin secretion in adult male and female rats. LHRH content in the preoptic-anterior hypothalamic (PO/AH) and midhypothalamic (MH) areas of neonatally estrogenized rats (1 mg on postpartum day 2) and of control male and female rats during the estrous cycle was determined by radioimmunoassay between 80 and 90 days of age. LHRH content was significantly greater in the PO/AH of neonatally estrogenized females than in estrogenized males whereas no differences in LHRH content of the PO/AH existed between control male and female rats. Neonatal administration of estrogen resulted in increased LHRH content in the MH of female rats and decreased LHRH content in male rats, as in the PO/AH; however, these differences were less marked in the MH and not statistically significant. The marked increase in serum LH concentration present in control cycling females in proestrus was abolished by neonatal estrogen treatment. Exposure to neonatal estrogen reduced FSH concentrations in males. The data are consistent with the concept that the hypothalamic-pituitary system is modified by estrogen circulating during the period of sexual differentiation. LHRH synthesis and release appear to be directly modified by neonatal estrogen. The effects of neonatal estrogen treatment in the PO/AH and MH areas may possibly involve two disparate types of alterations of developing LHRH neurons which modulate gonadotropin secretion in the adult rat.