Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H₂O₂) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca²⁺ increase of normal muscle cells is mediated by release of Ca²⁺ from intracellular stores, because it was not affected by incubation in Ca²⁺-free medium (CFM) containing 200 μM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca²⁺ increase, suggesting reduced Ca²⁺ release from intracellular stores. In normal Ca²⁺ medium, NKA and KCl caused normal Ca²⁺ signal in UC cells but reduced cell shortening. The decreased Ca²⁺ signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H₂O₂ scavenger catalase, suggesting involvement of H₂O₂ in UC-induced dysmotility. H₂O₂ levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H₂O₂ than normal cells, which were significantly reduced by catalase. H₂O₂ treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca²⁺ release observed in UC cells. In addition, H₂O₂ caused a measurable, direct release of Ca²⁺ from intracellular stores. We conclude that H₂O₂ may contribute to reduction of NKA-induced Ca²⁺ release from intracellular Ca²⁺ stores in UC and contribute to the observed colonic motor dysfunction.