Brainstem afferents to various nuclei of the basal forebrain of the rat were examined using the retrograde transport of wheat germ agglutinin-horseradish peroxidase. These forebrain nuclei included the medial septum-vertical limb of the diagonal band nucleus, the lateral septum, the nucleus of the horizontal limb of the diagonal band, the medial preoptic area and the magnocellular preoptic nucleus/substantia innominata. Medial septal-vertical limb of the diagonal band injections produced dense cell labeling in: raphe obscurus, nucleus incertus, central gray-pars alpha, locus coeruleus, raphe pontis, median raphe, nucleus of Darkschewitsch, a compact cell group within the mesencephalic gray dorsolateral to the nucleus of Darkschewitsch and the supramammillary nucleus. Lateral septal injections produced the heaviest cell labeling in the A1 and A2 areas (of Dahlstrom and Fuxe), the lateral parabrachial nucleus, the Kolliker-Fuse nucleus, the ventral tegmental area and the supramammillary nucleus. There were considerably fewer labeled cells overall with lateral septal as compared with medial septal injections. Brainstem projections to the horizontal limb of the diagonal band were pronounced. The most heavily labeled nuclei were A1, locus coeruleus, laterodorsalis (dorsolateral tegmental nucleus of Castaldi), raphe pontis, median raphe, lateral parabrachial nucleus, ventral tegmental area, nucleus of Darkschewitsch and the supramammillary nucleus. Medial preoptic area injections produced pronounced labeling in: A1 and A2 areas, raphe magnus, locus coeruleus, laterodorsalis, lateral parabrachial nucleus, pedunculopontine nucleus, peripenduncular nucleus and the supramammillary nucleus. The pattern of brainstem labeling obtained with magnocellular preoptic/substantia innominata injections was considerably different from the patterns seen with the other injections. Specifically, relatively few cell groups, essentially confined to the upper brainstem (rostral pons and midbrain), were densely labeled following magnocellular preoptic/substantia innominata injections. These included the medial parabrachial nucleus, the pedunculopontine nucleus, the dorsal raphe nucleus, the ventral tegmental area and the supramammillary nucleus. With the exception of the supramammillary nucleus, each of these cell groups was more heavily labeled with magnocellular preoptic/substantia innominata injections than with others of this series. The above describes the major brainstem projections to each of the forebrain sites.(ABSTRACT TRUNCATED AT 400 WORDS)