The present study has used a rat neuronal nitric oxide synthase (nNOS) antibody to examine the relationship of nNOS immunoreactivity to GnRH neurons in the ovariectomized and intact diestrous and proestrous rat. A striking band of nNOS-immunoreactive cells was identified in the rostral preoptic area which began in the median preoptic nucleus and organum vasculosum of the lamina terminalis and formed an inverted Y-type distribution above the rostral third ventricle at the level of the anteroventral periventricular nucleus. Another band of nNOS-immunoreactivity was found extending through the internal zone of the median eminence into the arcuate nucleus. Although nNOS immunoreactivity was not detected within GnRH neuronal cell bodies in any of the experimental groups, GnRH perikarya located in the rostral preoptic area, but not elsewhere, were found to be surrounded by nNOS-containing cells. In the median eminence, nNOS and GnRH immunoreactivities were distributed separately in the internal and external zones, respectively. These results provide evidence that, regardless of their pattern of activity, GnRH neurons in the female rat do not express nNOS. Instead, a close anatomical relationship between nNOS-immunoreactive cells and GnRH perikarya and fibers has been identified within specific sub-regions of the rostral preoptic area and in the median eminence. Such findings are compatible with a role for NO at both sites in regulating the release of GnRH throughout the estrous cycle.