In single cells, isolated by enzymatic digestion from the circular muscle layer of the lower esophageal sphincter (LES), acute experimental esophagitis (AE) alters signal transduction in response to a maximally effective dose of acetylcholine. In normal LES contraction was inhibited by M3 >> M1 or M2 antagonists. In AE inhibition by M2 antagonists increased significantly so that contraction was inhibited by M3 > M2 > M1 antagonists. In normal cells permeabilized by saponin, contraction was antagonized by antibodies against Gq/11, by the phosphatidylinositol-specific phospholipase C (PI-PLC) antagonist U 73122, but not by the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609, or by the phospholipase D pathway inhibitor propranolol. In AE contraction was reduced by Gq/11 and Gi3 antibodies and by U73122, propranolol and D609. After thapsigargin treatment of normal cells to reduce intracellular Ca++ stores, contraction was inhibited by M2 and M3 antagonists, by antibodies against Gq/11 and Gi3, by U73122, D609 and propranolol, suggesting that depletion of Ca++ stores reproduces the changes induced by AE. We conclude that in normal LES smooth muscle cells acetylcholine-induced contraction is mediated by M3 receptors linked to Gq/11 and PI-PLC, whereas in AE, contraction through this pathway is reduced, perhaps because of reduction in Ca++ stores, and a second pathway is activated by M2 receptors linked to Gi3, PC-PLC and phospholipase D.