Dopamine (DA) release in the medial preoptic area (MPOA) of the hypothalamus is an important facilitator of male sexual behavior. The presence of a receptive female increases extracellular DA in the MPOA, which increases further during copulation. However, the neurochemical events that mediate the increase of DA in the MPOA are not fully understood. Here we report that glutamate, reverse-dialyzed into the MPOA, increased extracellular DA, which returned to baseline after the glutamate was removed. This increase was prevented by co-administration of the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME), but not by the inactive isomer, Nw-nitro-d-arginine methyl ester (D-NAME). In contrast, extracellular concentrations of the major metabolites of DA were decreased by glutamate, suggesting that the DA transporter was inhibited. These decreases were also inhibited by L-NAME, but not D-NAME. These results indicate that glutamate enhances extracellular DA in the MPOA, at least in part, via nitric oxide activity. Therefore, glutamatergic stimulation of nitric oxide synthase may generate the female-induced increase in extracellular DA in the MPOA, which is important for the expression of male sexual behavior.