The striatum has nearly equal numbers of striatonigral and striatopallidal projection neurons. All are GABAergic and inhibitory, but they lie in separate neuronal circuits ('direct' and 'indirect', respectively) that appear to exert opposite effects on movement. Methods are needed to evaluate the function of each circuit. A potential way to control striatonigral neurons selectively is via M4 muscarinic receptors. The striatum has many more M4 receptors than other tissues, they are located on approximately half of all projection neurons, and mRNA for M4 receptors is prevalent only in striatonigral neurons. In order to more rigorously compare the distribution of M4 receptors on rat neurons in these pathways a toxin that binds with very high specificity to M4 receptors (m4-toxin) was biotinylated for use as a selective probe for M4 receptor protein. Pooled biotin-toxin complexes were found to retain high M4-specificity and affinity. Neurons were first labeled by retrograde transport of fluorescent microbeads (FluoSpheres) injected into the substantia nigra and globus pallidus. Coincident labeling of only 4% of the cells confirmed the validity of the retrograde labeling technique. Labeled neurons were probed for M4 receptor protein using biotinylated m4-toxin and fluorescent avidin. M4 receptors were found on 14% of indirect and 86% of direct neurons. It may be concluded that there is a relative abundance of M4 receptors controlling the direct pathway. This work supports the hypothesis that M4-selective drugs will prove useful to control the function of striatonigral neurons in the direct projection pathway.