We have previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect the release of intracellular Ca²⁺ stores. We therefore measured cytosolic Ca²⁺ levels in response to a maximally effective dose of ACh in Fura 2AM-loaded LES circular muscle cells and examined the contribution of H₂O₂ to the reduction in Ca²⁺ signal. In normal cells ACh-induced Ca²⁺ increase was the same in normal and Ca²⁺ free medium and was abolished by the PIP2-specific phospholipase C inhibitor U73122, confirming that the initial ACh-induced contraction depends on Ca²⁺ release from intracellular stores through production of IP3. In esophagitis, LES cells ACh induced Ca²⁺ increase was significantly lower in normal Ca²⁺ medium than in normal cells and was further reduced (~70%) when the cells were incubated in Ca²⁺ free medium. This reduction was partially reversed by the H₂O₂ scavenger catalase. Measurements of H₂O₂ in LES circular muscle showed significantly higher levels in esophagitis than in normal animals. When normal LES cells were incubated with H₂O₂, ACh-induced Ca²⁺ increase was significantly reduced both in normal Ca²⁺ and in Ca²⁺ free medium, and similar to those observed in the esophagitis animals. Similarly, the initial ACh-induced contraction was also reduced in normal cells incubated with H₂O₂. H₂O₂, when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca²⁺ signal in normal cells. In addition, H₂O₂-induced cell contraction was sensitive to depletion of stores by thapsigargin and, conversely, H₂O₂ reduced thapsigargin-induced contraction suggesting that thapsigargin and H₂O₂ may operate through similar mechanisms. Measurements of Ca2⁺-ATPase activity indicate that both H₂O₂ and thapsigargin a caused a reduction in Ca2⁺-ATPase activity, confirming similarity of mechanism of action. We conclude that H₂O₂ may be at least partly responsible for impairment of Ca²⁺ release in acute experimental esophagitis by inhibiting Ca²⁺uptake and refilling of Ca²⁺ stores.