The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described. We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus. These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents. These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)