Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase-anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance P-immunoreactive sites were first localized using 3,3'-diaminobenzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.