Striatal m4 muscarinic receptors are important because their blockade controls movement, and they are preferentially located on striatal neurons that project to the internal globus pallidus. The following studies were performed in vitro to provide a basis for using antimuscarinic toxins to study the effects of selective m4 blockade on movement in vivo. Because m4-toxin has limited selectivity alone (102-fold higher affinity for m4 than m1 receptors), m1-toxin was used first to occlude m1 receptors selectively, fully and irreversibly. It blocked 42% of the sites for 1.0 nM 3H-N-methylscopolamine in rat striatal membranes and 43% in sections of cat striatum. m4-Toxin (>500-fold higher affinity for m4 than m2, m3 or m5 receptors) blocked 88% of the residual, non-m1 sites in membranes, showing 64 pmol m4 receptors/g tissue. In comparison, AFDX-116, biperiden, clozapine, gallamine, hexahydrodifenidol, himbacine, R(+)hyoscyamine, methoctramine, pirenzepine, silahexocyclium, trihexyphenidyl and tripitramine did not distinguish m4 from other non-m1 receptors. 3H-Pirenzepine dissociated twice as rapidly from non-m1 as m1 receptors. Autoradiography was used to test the idea that m4 receptors are localized preferentially in the striosomes of the cat striatum. Non-m1 receptors were distributed equally in striosomes and matrix, indicating that striatal neurons with m4 receptors are in both compartments. Thus m1-toxin facilitates studies of m4 receptors by occluding m1 receptors, and m4-toxin is a selective antagonist for residual m4 receptors.