A surge of prolactin (PRL) occurs in female rats during proestrus in response to elevated estradiol and progesterone levels. Dopamine is the primary hypothalamic inhibitor of PRL secretion from the pituitary. Using double-label immunocytochemistry, we investigated the pattern of estrogen receptor-alpha (ER-alpha) immunoreactivity in dopaminergic neurons in the arcuate nucleus (ARC) and the periventricular nucleus (PeVN) during the proestrous PRL surge and compared it to that during diestrus, when PRL levels are constantly low. Our results showed that during diestrus >80% of dopaminergic neurons in the ARC were also positive for ER-alpha, and this colocalization percentage decreased significantly during proestrus. By contrast, <15% of dopaminergic neurons in the PeVN expressed ER-alpha, and the low percentage of ER-alpha expression was unchanged throughout proestrus and diestrus. Results from estrogen plus progesterone treated ovariectomized rats showed similar patterns of ER-alpha expression within the ARC and the PeVN and, once again, compared with the control group, had a significant reduction in ER-alpha immunoreactivity in dopaminergic neurons in the ARC, but not in the PeVN. These results provide an anatomic basis that dopaminergic neurons in the ARC and the PeVN are functionally different regarding to ER-alpha expression. Our study also supports the hypothesis that dopaminergic neurons in the ARC are an important neuronal population responsive to estrogen by changing the expression of ER-alpha in those neurons. This modification of sensitivity of dopaminergic neurons in the ARC in response to ovarian steroids may be an important molecular mechanism involved in PRL regulation, including the regulation of the proestrous surge of PRL.