The effect of long-term decreases in circulating concentrations of prolactin was determined on the responsiveness of tuberoinfundibular dopamine (DA) neurons to this hormone. The activity of these neurons in ovariectomized rats was estimated by measuring the rate of DA synthesis (DOPA accumulation after the administration of a decarboxylase inhibitor) in the median eminence at various times after serum concentrations of prolactin had been reduced by hypophysectomy or the chronic administration of a DA agonist (bromocriptine, 3 mg/kg/day). The concentration of DA in the median eminence, but not in striatum, declined progressively up to 12 days after hypophysectomy, but did not change at any time during bromocriptine treatment. On the other hand, norepinephrine concentrations in the median eminence were increased 12 days after both treatments. Within 24 h after hypophysectomy or the first injection of bromocriptine the rate of DA synthesis in the median eminence was decreased; this decrease was maintained for at least 12 days suggesting that tuberoinfundibular DA neuronal activity is normally maintained by endogenous prolactin. Intracerebroventricular (ICV) injections of prolactin (10 µg, 12 h prior to sacrifice) increased the rate of DA synthesis in the median eminence of control, 24-hour hypophysectomized and 24-hour bromocriptine-treated rats. After longer periods (6–12 days) of bromocriptine treatment or after hypophysectomy the responsiveness of tuberoinfundibular DA neurons to prolactin was reduced. Dose-response studies revealed that the sensitivity and magnitude of response to ICV prolactin was markedly reduced in 12-day hypophysectomized rats. The decreased responsiveness of tuberoinfundibular DA neurons to the actions of prolactin following long-term bromocriptine treatment and hypophysectomy is most likely due to the prolonged decrease in circulating prolactin, since the concurrent daily administration of bromocriptine and ICV prolactin for 12 days maintained the ability of tuberoinfundibular DA neurons to respond to prolactin. The decreased basal activity and responsiveness of tuberoinfundibular DA neurons to prolactin induced by hypoprolactinemia is reversible; 12 days after terminating chronic bromocriptine treatment basal activity and the responsiveness of tuberoinfundibular DA neurons to prolactin return to control values. These results suggest that the response of tuberoinfundibular DA neurons to prolactin is determined, in part, by the preceding circulating levels of this hormo