Supraspinal afferents to the pontine micturition center, Barrington's nucleus, were investigated in the rat by visualization of the retrograde tracer, cholera-toxin subunit B, in neurons following iontophoretic injection into Barrington's nucleus. Tissue sections from five rats with injections primarily localized in Barrington's nucleus revealed numerous retrogradely labeled neurons throughout all rostrocaudal levels of the periaqueductal gray (particularly its ventrolateral division), in the lateral hypothalamic area (particularly medial to the fornix), and in the medial preoptic nucleus. Retrogradely labeled neurons were also consistently found in the nucleus of the solitary tract, in the vicinity of the lateral reticular nucleus, nucleus paragigantocellularis, parabrachial nucleus, Kölliker-Fuse nucleus, cuneiform nucleus, raphe nucleus and zona incerta. In the hypothalamus, in addition to the perifornical region, retrogradely labeled neurons were found in all cases in the tuberomammillary nucleus, premammillary nucleus, dorsal hypothalamic area, ventromedial hypothalamic nucleus, and the paraventricular nucleus. At more rostral levels, in addition to the medial preoptic area, retrogradely labeled neurons were seen in the bed nucleus of the stria terminalis and in a region just lateral to the supraoptic nucleus near the medial amygdaloid nucleus. Retrogradely labeled neurons were also observed in the motor, insular, and infralimbic cortices. Injections of anterograde tracers (cholera-toxin subunit B or Phaseolus vulgaris leucoagglutinin) into the Kölliker-Fuse nucleus, the ventrolateral periaqueductal gray, lateral hypothalamic area, or medial preoptic area, resulted in fiber labeling within Barrington's nucleus, confirming the retrograde tracing studies. As previously reported, numerous neurons in Barrington's nucleus were immunoreactive for corticotropin-releasing hormone. Double-labeling studies revealed afferent fibers from the periaqueductal gray and lateral hypothalamic area overlapping the corticotropin-releasing hormone-immunoreactive neurons of Barrington's nucleus, and in some cases anterogradely labeled fibers with varcosities appeared to target these neurons. The present results suggest that Barrington's nucleus in the rat receives neuronal inputs from brainstem nuclei as well as from forebrain limbic structures including hypothalamic nuclei, the medial preoptic nucleus, and cortical areas involved in fluid balance or blood pressure regulation. In light of the role of Barrington's nucleus in micturition, the integration of these various inputs may be important for co-ordinating urinary function with fluid and cardiovascular homeostasis. Additionally, as neurons in Barrington's nucleus are immunoreactive for the stress-related neurohormone, corticotropin-releasing hormone, these diverse inputs may regulate stress-related functions of this nucleus.