In many vertebrate species the medial preoptic area projects to a premotor nucleus, the periaqueductal central gray (PAG). This connection plays an important role in the control of reproductive behavior. In male Japanese quail (Coturnix japonica) specifically, the medial preoptic nucleus (POM), where various types of sensory inputs converge, is a critical site for the activational action of testosterone on male sexual behavior. To activate male copulatory behavior, testosterone must be aromatized to estradiol within the POM and aromatase-immunoreactive cells in the POM are the main source of projections to the PAG. The POM–PAG connection is thus an important functional circuit integrating the sensory with premotor components of sexual behavior. Contrary to what is observed in males, testosterone does not activate male-typical copulatory behavior in females and we investigated here via retrograde tracing methods whether this behavioral sexual difference is associated with a sex difference in connectivity between POM and PAG. Fluorescent microspheres were injected in the PAG of male and female quail and retrogradely labeled fluorescent cells counted in four fields of the POM in sections that had been immunolabeled for aromatase. Males had more aromatase-immunoreactive neurons projecting to the PAG than females and this difference was most prominent in the caudolateral part of the nucleus that has been specifically implicated in the control of male copulatory behavior. These data therefore support the hypothesis that sex differences in POM–PAG connectivity are causally linked to the sex difference in the behavioral response to testosterone. J. Comp. Neurol. 500:894–907, 2007. © 2006 Wiley-Liss, Inc.