The rostro-caudal distribution of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of neonatally estrogenized (10 micrograms EB for 1-5 days), sterile female rats was compared with that of cycling rats at 50, 100 and 240 days of age. Serial slices of 400 microns in thickness in total were cut from a hypothalamic block, and the LHRH content of each slice was measured by radioimmunoassay. Two peaks of LHRH in the rostro-caudal direction were apparent in all groups. The rostral peak corresponded to the LHRH-producing cells in the septal-preoptic region and storage of LHRH in the organum vasculosum lamina terminalis (OVLT), while the caudal peak corresponded to that of LHRH in the median eminence. Thus, the total LHRH content of the regions of the hypothalamus was calculated separately either anterior or posterior to the suprachiasmatic nucleus, which were designated as the rostral hypothalamus (RH) and mediobasal hypothalamus (MBH), respectively. The total content of LHRH in the 50-day-old sterile rats was significantly higher than that in the estrous females in both RH and MBH. This was also true for the MBH of 240-day-old sterile females in comparison to that of estrous females but not so in 100-day-old females. The present results suggest that sterility in neonatally estrogenized rats is not due to a reduction in LHRH content of the hypothalamus but, rather, is due to changes in LHRH-releasing systems. Furthermore, in the control females, ovariectomy performed at 22 days of age induced a striking decrease in LHRH content in the MBH at 50 days of age, while it remained unchanged after ovariectomy in the neonatally estrogenized sterile rats. These findings suggest that the hypothalamic mechanism involved in the release of LHRH after ovariectomy was damaged by neonatal treatment with estrogen.