Impairment in gallbladder emptying, increase in residual volume, and reduced smooth muscle contractility are hallmarks of acute acalculous cholecystitis and seem to be related to ischemia/reperfusion (I/R). This study was designed to determine the effects of tempol, a general antioxidant, on I/R-induced changes in gallbladder contractile capacity, the mechanisms involved in the contractile process, and the level of inflammatory mediators. Experimental gallbladder I/R was induced in male guinea pigs by common bile duct ligation for 2 days, then a deligation of the duct was performed and after 2 days the animals were sacrificed. A group of animals was treated with tempol, administered in the drinking water at 1 mmol/l for 10 days prior the bile duct ligation and until animal sacrifice. Isometric tension recordings showed that KCl and cholecystokinin-induced contractions were impaired by I/R, which correlated with decreased F-actin content and detrimental effects on Ca(2+) influx. In addition, I/R depolarized mitochondrial membrane potential, as indicated by the reduction of the heterogeneity of the rhodamine123 fluorescence signal, and increased the expression of NF-kappaB, COX-2, and iNOS. Tempol treatment improved contractility via normalization of Ca(2+) handling and improvement of F-actin content. Moreover, the antioxidant ameliorated mitochondrial polarity and normalized the expression levels of the inflammatory mediators. These results show that antioxidant treatment protects the gallbladder from I/R, indicating the potential therapeutic benefits of tempol in I/R injury.