Ovariectomized rats, when transplanted with 4 anterior pituitaries (APs) to the kidney capsule for 2–3 weeks, had elevated plasma prolactin (PRL) levels (3.8-fold) and showed decreased in situ AP weights (0.62-fold) and PRL concentrations (0.63-fold). The concentrations of dopamine (DA) and oxytocin (OT) in pituitary portal plasma of hyperprolactinemic rats were increased 1.7- and 1.9-fold, respectively. However, the levels of vasoactive intestinal peptide (VIP) in pituitary portal plasma of these rats were decreased 0.31-fold. The secretion of DA, dihydroxyphenylalanine (DOPA) and OT from fetal hypothalamic cells in primary culture was increased, whereas VIP secretion from these cells was reduced in a dose-dependent fashion following PRL treatment. These data are the first in vivo and in vitro demonstration of a stimulatory action of PRL on OT release and an inhibitory action of PRL on VIP release. Furthermore, these data suggest that a subtle imbalance between the secretion of the PRL-inhibiting factor (DA) and the PRL-releasing factors (VIP and OT) during elevated systemic levels of PRL is responsible for decreased lactotrophic function.