Estradiol stimulates the synthesis and secretion of PRL and lactotroph proliferation, and its long-term administration induces PRL-secreting pituitary tumors. We examined changes in the number of proliferating lactotrophs throughout the estrous cycle in female rats and the involvement of the brain in the regulation of the lactotroph proliferation by anesthetizing with pentobarbital. Female rats revealing regular 4-day estrous cycles were injected ip with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) 3 h before decapitation to label DNA-replicating cells. Dispersed anterior pituitary cells obtained from these rats were stained for PRL and BrdU with a double labeling immunofluorescence technique. The rate of lactotroph proliferation was represented by the BrdU-labeling index (BrdU-labeled lactotrophs as a percentage of total lactotrophs counted). Lactotrophs from rats injected with BrdU at 1000 h showed a high BrdU-labeling index of 2.6% on estrus, whereas they showed almost undetectable levels of the BrdU-labeling index during the other stages of the estrous cycle. The anterior pituitary cells other than lactotrophs were scarcely labeled during any stages. The BrdU-labeling index began to rise by the midnight between proestrus and estrus, peaked between 0800 and 1200 h, and returned to undetectable levels by the midnight between estrus and diestrus I. Pentobarbital (35 mg/kg, i.p.) injected at 1345 h on proestrus, which was effective in blocking ovulation on estrus, eliminated completely the increase in the BrdU-labeling index as determined by BrdU injection at 1000 h on estrus. In contrast, the high BrdU-labeling index on estrus was partly suppressed to a level of 1.4% by pentobarbital injection at 0900 or 2100 h on proestrus. The rats injected with pentobarbital at 1345 h on proestrus did not show any increases in the BrdU-labeling index even after BrdU injection was delayed from 1000 to 1400 h on estrus. However, a high BrdU-labeling index of 3.7% was obtained in these animals when BrdU was injected at 1000 h on diestrus I. We conclude that 1) lactotrophs of cycling female rats proliferate selectively on the day of estrus; and 2) the proliferation of lactotrophs on estrus is not due to a direct action on the anterior pituitary of estradiol secreted from the ovaries but triggered by neural events occurring during the proestrous afternoon, which are closely related with the regulation of preovulatory surges of gonadotropin and PRL secretion.