Five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified by molecular cloning and designated m(1)-m(5), of which four correspond to the functional subtypes M(1), M(2), M(3), and M(4) in primary tissues. The presence of M(5) receptors in tissues remains uncertain. The present study was designed to explore the diversity and cellular distribution of various mAChR subtypes in human hearts. Competition binding of [N-methyl-(3)H]-scopolamine methyl chloride with various mAChR antagonists yielded data consistent with the presence of multiple subtypes (M(1)/M(2)/M(3)/M(5)) of mAChRs in both human atrial (HA) and ventricular (HV) tissues. Expression of mRNAs encoding all five subtypes was readily detected by reverse transcription-polymerase chain reaction in both HA and HV samples. Immunoblotting with subtype-specific antibodies confirmed the presence of M(1), M(2), M(3), and M(5), but not M(4), proteins in membrane preparations from both HA and HV. The protein levels of M(1) and M(2) were comparable between HA and HV. Although the density of M(3) appeared approximately 10-fold higher in HV than HA, that of M(5) was approximately 5 times lower in HV than in HA. Positive immunostaining of single ventricular myocytes by M(1), M(2), M(3), and M(5) antibodies, respectively, was consistently detected. Under confocal microscopy, M(5) showed characteristic localization to the intercalated discs, whereas other subtypes were more evenly distributed throughout the surface membrane. Our results provide the first molecular evidence for the presence of multiple subtypes of mAChR, including endogenous M(5) receptors, in human hearts and suggest that different subtypes have different tissue distributions and cellular localization.