The binding of [3H]5-hydroxytryptamine ([3H]5-HT) to cortical membranes was examined in female rats during diestrus, proestrus and estrus. Serotonin binding was lowest during the early afternoon of proestrus and highest during the afternoon of estrus with diestrous values in between. The high estrous values were associated with a significant increase in Bmax and a decrease in Kd. However, the increase in binding actually took place during the late afternoon of proestrus, when there was a rapid increase in binding from noon to 18.00 h. Binding remained at this elevated level throughout the next day. There were no changes in the binding of [3H]ketanserin or [3H]spiroperidol to cortical membranes during the estrous cycle, so the differential binding of [3H]5-HT is most likely the result of variations in 5-HT1 rather than 5-HT2 receptors. These observations of changes in serotonin binding in a brain area nearly devoid of sex steroid receptors suggest that the hormonal fluctuations accompanying the female estrous cycle influence brain areas other than those classically thought to regulate neuroendocrine function.