In heart tissue five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified, designated m1-m5, of which only M1, M2 and M3 have functional evidences for their role in cardiac physiology. The present study was designed to explore the diversity of mAChR subtypes in human hearts and determine whether these subtypes are able to interact themselves. Expression of mRNAs encoding all five subtypes was readily detected by RT-PCR reaction in both atrial (A) and ventricle (V) samples. Immunoblotting, MABA and ELISA with subtype-specific antibodies confirmed the presence of M1, M2, M3, M4 and M5 proteins in membrane preparations from both A and V. Kinetic characterization using [(3)H]-QNB shown: (1) atrium had greater B(max) than did the ventricle, (2) [(3)H]-QNB behave as an allosteric modulator, inducing cooperativity at high and disclosing heterogeneity at low concentrations, (3) heterogenity was observed in pirenzepine, biperiden and tropicamide competition curves, being the high affinity sites compatible with M1 and M4 muscarinic receptor subtypes and (4) methoctramine competition curves in presence of selective muscarinic receptor subtypes antagonist displayed heterogeneity profile still maintaining cooperativity (n(H)>1), indicating muscarinic receptors subtypes are able to form homo- and hetero-oligomers. In conclusion, our results provide molecular and kinetic evidence for the presence of multiple subtypes of mAChR in human hearts, which are able to undergo discrete transitions from a non-cooperative kinetics of non-interacting monomers to a cooperative kinetics of interacting oligomers.