Long-term 17β-estradiol (E2) treatment in rats decreases tuberoinfundibular dopaminergic (TIDA) neuronal function. The objective of this study was to determine if the decline in TIDA function after E2 treatment in Fischer 344 (F344) rats is sustained long after removal of E2. Ovariectomized (OVX) F344 rats were each implanted with an E2-containing or empty Silastic capsule for 4 weeks; the capsule was then removed, and 26 weeks later acute experiments were performed. Release of 3H from median eminence tissue in vitro in response to electrical stimulation after 3H-DA accumulation was not different between E2-treated rats and OVX controls, even though serum prolactin (PRL) was 4-fold greater in E2-treated animals. Acute administration of apomorphine hydrochloride, a DA receptor agonist, at 2 doses, reduced serum PRL values as much in E2-treated animals as in OVX control rats. Injection of morphine sulfate or nomifensine maleate, which directly influence TIDA neurons, resulted in nonsignificant serum PRL responses in animals long after E2 withdrawal as compared to the greater response in OVX control rats. To further evaluate TIDA neuronal function, OVX non-E2-treated rats and animals 26 weeks after E2 withdrawal received a 3-day E2 challenge which increased the stimulation-evoked release of 3H from the median eminence tissue in vitro 2-fold in OVX control rats but had no effect in rats given E2 26 weeks previously. The difference in the stimulation-evoked release occurred in the presence of similar circulating serum PRL levels in the two groups as a result of the 3-day E2 treatment. These data indicate that TIDA neurons exhibit a decreased responsiveness to most stimuli after long-term withdrawal from chronic E2 treatment, even though the E2-treated anterior pituitary was fully responsive to the dopaminergic agonist, apomorphine. Thus, E2 administration to OVX F344 rats for 4 weeks appears to result in a permanent decline in TIDA neuronal function. The mechanism(s) responsible for this permanent effect of E2 is not known at present