The distribution of cholinergic interneurons with respect to enkephalin-rich patches in the caudate nucleus of the cat was examined using both computer-assisted 3-D reconstruction and immunocytochemical techniques. Examination of the 3-D distribution of perikarya staining for choline acetyltransferase (ChAT) revealed that these cells were not evenly distributed within the caudate nucleus but exhibited areas of increased and decreased density. Comparison of the 3-D distribution of cholinergic perikarya to that of the enkephalin-rich patches indicated that areas of increased ChAT+ cell density often corresponded to the positions of enkephalin-rich patches within the dorsal-lateral caudate nucleus. At more ventral regions, there was no clear correspondence between areas of increased ChAT+ cell density and enkephalin-rich patches. In agreement with these observations, a quantitative analysis of sections double-labeled for ChAT and enkephalin revealed that the density of cholinergic neurons within enkephalin-rich patches was twice that in the surrounding tissue in the dorsal region of the caudate nucleus. In contrast at more ventral levels, the difference in the density of ChAT+ cells in enkephalin-rich patches did not significantly differ from that in the surrounding striatal tissue. Both the results of the 3-D and the double-labeling analysis suggest that cholinergic neurons are not evenly distributed within the caudate nucleus of the cat but form loose clusters which are associated dorsally with the enkephalin-rich patches. These results also provide further evidence of heterogeneity within the striosomal compartment in the cat.