The autoradiographic distribution of M1 and M2 muscarinic cholinergic binding sites was studied in the striatum of the cat, monkey, and human, and concurrent binding assays were carried out on striatal tissue sections from the cat. M1 sites were directly labeled with 3H-pirenzepine; M2 sites were labeled as a consequence of binding competition between pirenzepine and 3H-N-methylscopolamine. Serial section analysis with autoradiograms and stained tissue sections allowed for comparisons among M1 and M2 binding distributions and AChE staining patterns. The 2 subtypes of binding sites demonstrated distinct striatal distributions. M2 sites were virtually homogeneous except in the ventral striatum, where zones of sparse and especially dense binding were observed. Striatal M1 sites were generally more abundant than M2 sites and showed similar heterogeneity in the ventral striatum. Dorsally, however, patches of dense M1 binding were found, and proved to correspond with AChE-poor striosomes, hallmarks of striatal compartmentalization. The finding of differing distributions for the 2 subtypes of muscarinic cholinergic binding sites suggests a mechanism for the intrinsic spatial segregation of striatal cholinergic function. Further, the striosomal patterning of M1 binding indicates that certain aspects of cholinergic function in the striatum may be constrained and thus regulated by the compartmental ordering characteristic of this region of the basal ganglia.