Reactive oxygen species (ROS) have been implicated in the pathogenesis of muscle dysfunction in acute inflammatory processes. The aim of these studies was to determine the effects of ROS on gallbladder muscle function in vitro. Single muscle cells were obtained by enzymatic digestion. H₂O₂ (70 µM) caused maximal contraction of up to 14% and blocked the response to CCK-8, ACh, and KCl. It did not affect the contractions induced by guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and inositol 1,4,5-trisphosphate that circumvent membrane receptors. The contraction induced by H₂O₂ was inhibited by AACOCF₃ [cytosolic phospholipase A₂ (cPLA₂) inhibitor], indomethacin (cyclooxygenase inhibitor), chelerythrine [protein kinase C (PKC) inhibitor], or PD-98059 [mitogen-activated protein kinase (MAPK) inhibitor]. H₂O₂ also reduced the CCK receptor binding capacity from 0.36 ± 0.05 pmol/mg protein (controls) to 0.17 ± 0.03 pmol/mg protein. The level of lipid peroxidation as well as the PGE₂ content was significantly increased after H₂O₂ pretreatment. Unlike superoxide dismutase, the free radical scavenger catalase prevented the H₂O₂ induced contraction, and its inhibition of the CCK-8 induced contraction. It is concluded that ROS cause damage to the plasma membrane of the gallbladder muscle and contraction through the generation of PGE₂ induced by cPLA₂-cyclooxygenase and probably mediated by the PKC-MAPK pathway.