Studies using estrogen receptor alpha(ER) knock-out mice indicate that ER alpha masculinizes male behavior. Recent studies of ER alpha and male prosocial behavior have shown an inverse relationship between ER alpha expression in regions of the brain that regulate social behavior, including the medial amygdala (MeA), and the expression of male prosocial behavior. These studies have lead to the hypothesis that low levels of ER alpha are necessary to "permit" the expression of high levels of male prosocial behavior. To test this, viral vectors were used to enhance ER alpha in male prairie voles (Microtus ochrogaster), which display high levels of prosocial behavior and low levels of MeA ER alpha. Adult male prairie voles were transfected with ER alpha in the MeA (MeA-ER alpha) or the caudate-putamen (ER alpha control) or luciferase (MeAsitespecific control), and 3 weeks later tested for spontaneous alloparental behavior and partner preference. Enhancing ER alpha in the MeA altered/reduced male prosocial behavior. Only one-third of MeA-ER alpha males, compared with all control males, were alloparental. Me-A-ER alpha males also displayed a significant preference for a novel female. This is a critical finding because the manipulations of neuropeptides, oxytocin and vasopressin, can inhibit the formation of a partner preference, but do not lead to the formation of a preference for a novel female. The results support the hypothesis that low levels of ER alpha are necessary for high levels of male prosocial behavior, and provide the first direct evidence that site-specific ER alpha expression plays a critical role in the expression of male prosocial behavior.