Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H₂O₂ production, lipid peroxidation, and increased PGE₂ synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A₂ (cPLA₂), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H₂O₂ via activation of NADPH and xanthine oxidase. H₂O₂ causes lipid peroxidation and activates cPLA₂ to increase PGE₂ production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.