Background and Aim: Manometric studies on the human lower esophageal sphincter (LES) have shown radial asymmetry of the high-pressure zone (HPZ). The aim of this study was to compare the functional properties of human LES clasp and sling muscles, and to look at their relationship with the expression of muscarinic receptors and intracellular Ca(2+) concentration. Methods: Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to acetylcholine were studied. Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression of five subtypes of muscarinic receptors. Intracellular Ca(2+) ([Ca(2+)]i) was measured using laser scanning confocal microscopy. Results: Acetylcholine caused a concentration-dependent increase in the tension of sling and clasp strips, the sling strip being stronger than clasp (P = 0.00). Messenger RNA and protein for the five muscarinic acetylcholine receptor (mAChRs) expressed in the sling and clasp muscles were highest for M2, and then in decreasing levels: M(3) > M(1) > M(4) > M(5). Acetylcholine caused significant elevation of [Ca(2+)]i in sling and clasp muscle cells in the presence of extracellular Ca(2+) (1.5 mmol/L), and Ach-induced [Ca(2+)]i elevation was 1.6 times greater in sling cells than in clasp cells. Conclusion: Variation of intracellular concentrations of Ca(2+) may be the reason for differential responses to acetylcholine for sling versus clasp fibers. However, these differences are not associated with the distribution and the level of expression of the five mAChRs between the two muscle types. Further study should focus on the ligand affinity and signal transduction pathway.