The supraspinal regulation of genital reflexes is poorly understood. The brainstem nucleus paragigantocellularis (nPGi) of rats is a well-established source of tonic inhibition of genital reflexes. However, the organization, gonadal steroid receptor expression, and activity of nPGi afferents during sex have not been fully characterized in male and female rats. To delineate the anatomical and physiological organization of nPGi afferents, the retrograde tracer Fluoro-Gold (FG) was injected into the nPGi of sexually experienced male and female rats. Animals engaged in sexual behavior 1 hour before sacrifice. Cells containing FG, estrogen receptor-α (ERα), androgen receptor (AR), and the immediate-early gene product Fos were identified immunocytochemically. Retrograde labeling from the nPGi was prominent in the bed nucleus of the stria terminalis, paraventricular nucleus (PVN), posterior hypothalamus, precommissural nucleus, deep mesencephalic nucleus, and periaqueductal gray (PAG) of both sexes. Sex differences were observed in the caudal medial preoptic area (MPO), with significantly more FG+ cells observed in males, and in the PAG and inferior colliculus, where significantly more FG+ cells were observed in females. The majority of regions that contained FG+ cells also contained ERα or AR, indicating sensitivity to gonadal steroids. The proportions of FG+ cells that co-localized with sex-induced Fos was high in the PVN of both sexes and high in the MPO of males but low in the PAG of both sexes despite the large number of PAG-nPGi output neurons and Fos+ cells in both sexes. The characterization of these afferents will lead to a further understanding of the neural regulation of genital reflexes. J. Comp. Neurol. 508:771–794, 2008. © 2008 Wiley-Liss, Inc.