Maternally behaving virgin rats are capable of releasing prolactin reflexively in response to stimulation by pups, especially during the proestrous/estrous phase of the cycle. When such rats are chronically exposed to pups they usually undergo a state of pseudopregnancy during which prolactin is secreted in a pattern of bocturnal surges. The present series of experiments evaluated (a) the initiation of nocturnal prolactin surges in maternally behaving virgins, (b) the role of estrogen in the reflexive release of prolactin, and (c) the influence of gender on these two modes of prolactin secretion. It was found that the nocturnal surges of prolactin are already present on Days 1 and 2 of pup-induced pseudopregnancy. At this stage, however, the surges are not yet autonomous, seeing that pseudopregnancy is interrupted shortly after removal of the pups on Day 2. Activation by vaginocervical stimulation of the "mnemonic" neurogenic system that controls the autonomous nocturnal prolactin surges did not interfere with the reflexive pup-induced release of prolactin in maternally behaving virgins. The capacity of reflexive prolactin release in the virgin rat was abolished by ovariectomy, restored by estrogen replacement, and persisted for only 24 hr following estrogen removal. Paternally behaving males subjected to chronic exposure to pups were incapable of secreting nocturnal surges of prolactin characteristic of the pseudopregnant female. Such males were also incapable of releasing prolactin reflexively in response to stimulation by pups, even when supplemented with exogenous estrogen. These results indicate that the two modes of prolactin secretion are sex dependent, and that the maternally behaving virgin, unlike the postpartum rat, requires concurrent estrogenic facilitation for releasing prolactin in response to stimulation by young.