A family of muscarinic ACh receptor genes are expressed in hippocampus, but little is known about the localization of the encoded proteins and their regulation by cholinergic innervation. Subtype-specific antibodies were used to localize m1-m4 proteins in the hippocampal formation by immunocytochemistry and to determine the alterations in the subtypes following deafferentation. Each of the receptors is differentially localized in Ammon's horn and dentate gyrus, with highly complementary distributions. m1 is widely expressed in somata and dendrites of pyramidal neurons and granule cells in dentate gyrus. m2 immunoreactivity is expressed mostly in nonpyramidal neurons, and in several discrete bands of fibers and puncta surrounding pyramidal neurons and other layers. m3 is enriched in pyramidal neurons, the neuropil in stratum lacunosum-moleculare and the outer third of the molecular layer of dentate gyrus. m4 is enriched in nonpyramidal neurons, in fiber pathways (alveus, fimbria, and hippocampal commissure), and in the inner third of the molecular layer. Fimbria-fornix lesions decreased ipsilateral m2- and m4-immunoreactive axons in the fimbria, with no apparent changes in the distribution of any of the receptors in hippocampus. 192-IgG immunotoxin lesions of the cholinergic septohippocampal projections, which spare noncholinergic projections, produced a small decrease in m2-immunoreactive fibers in the fimbria with no other major changes in the distribution of subtypes. Immunoprecipitation studies at 3-28 d following fimbria-fornix lesions revealed a 25% loss of m2 at 3 d in hippocampus, and upregulation of both m1 (20-29% at 7-14 d) and m4 (44% at 28 d). Thus, the vast majority of muscarinic receptor subtypes are intrinsic to the hippocampal formation and/or nonseptal hippocampal afferents. A subset of m2 and m4 are presynaptically localized, with m2 in cholinergic axons and m2 and m4 possibly in noncholinergic axons that comprise the septohippocampal pathway. The unique laminar and regional distributions of m1-m4 in the hippocampus reflect differential cellular and subcellular distributions of the subtypes and/or selective association of receptor subtypes with certain afferent and intrinsic connections. These results indicate that each subtype likely has a different role in cholinergic modulation of excitatory and inhibitory hippocampal circuits.