The present study addressed the hypothesis that differential localization of ml-m4 subtypes in the inner one-third of the dentate molecular layer is due to selective presynaptic expression of the receptor proteins on the hippocampal commissural/associational pathways. Physical and chemical lesions of the commissural and associational pathways were used to denervate afferent terminals in the inner one-third of the molecular layer, and fluid injections were used to lesion granule cells, their postsynaptic target. Immunocytochemistry utilizing muscarine acetylcholine receptor (mAChR) subtype-specific antibodies was used to identify changes in expression patterns in the molecular layer postlesion. m1 immunoreactivity in the molecular layer did not change after commissural/associational pathway lesions. m2 immunoreactivity in the inner one-third of the molecular layer was attenuated only after lesions involving the associational pathway. In contrast, m3 and m4 immunoreactivity in the inner one-third of the molecular layer was almost completely abolished by lesions of both pathways simultaneously. Granule cell lesions greatly attenuated m1 and m3 immunoreactivity in the molecular layer, with little to no diminution of m2 and m4 immunoreactivity. The results indicate that, in the inner one-third of the molecular layer, m1 and m3 are mainly postsynaptic on granule cells, whereas m2 and m4 are presynaptic on the commissural/associational pathways. This study provides direct anatomical evidence for the diversity of molecular subtypes presynaptically on the commissural/associational pathways.