The distribution of cells stained immunocytochemically for the cholinergic marker choline acetyltransferase was compared to the pattern of substance P immunoreactivity in the caudate nucleus of adult cats using a double-label immunocytochemical protocol and three-dimensional reconstructions of adjacent sections single-labeled for either substance P or choline acetyltransferase. Substance P immunoreactivity was distributed in a highly complex mosaic within the caudate nucleus of the cat. In the dorsal caudate nucleus, substance P-rich zones consisting of either clusters of substance P-positive cell bodies or fibers were seen against a lighter staining background. The density of cholinergic neurons was found to be significantly greater within these substance P-rich patches in comparison to surrounding regions. The pattern of substance P immunoreactivity within the ventral caudate nucleus differed from that in more dorsal regions. Clear substance P-rich patches were not seen in this region, but a large substance P-rich area consisting of a dense plexus of substance P-containing fibers was visible. Embedded within this substance P-rich area were fairly discrete patches of light substance P staining. As in the dorsal caudate nucleus, increased numbers of cholinergic neurons and processes were associated with substance P-rich regions in the ventral caudate nucleus. Choline acetyltransferase-positive perikarya also appeared to be concentrated in substance P-rich areas in the nucleus accumbens and olfactory tubercle. The results of this study suggest that a close relationship exists between the distribution of substance P fibers and cholinergic perikarya in the striatum of the cat.