Loss of gastrointestinal (GI) barrier integrity has been implicated in a wide range of inflammatory illnesses, including alcoholic cirrhosis. Using monolayers of Caco-2 (intestinal) cells as a model, we showed that the ability of ethanol (EtOH) to disrupt intestinal barrier integrity depends on damage to the microtubule (MT) cytoskeleton, especially oxidative injury. One drug that prevented both the MT damage and barrier disruption was L-N(6)-1-iminoethyl-lysine, a selective inhibitor of the inducible form of nitric-oxide synthase (iNOS). Because of this finding and because overproduction of nitric oxide (NO) and generation of peroxynitrite (ONOO(-)) have been proposed to be responsible for mucosal injury in other GI disorders, we sought to determine whether NO overproduction and ONOO(-) formation mediates EtOH-induced MT damage and loss of intestinal barrier function. To this end, Caco-2 monolayers were exposed to EtOH or to authentic ONOO(-) or ONOO(-) generators with or without pretreatment with iNOS inhibitors or antioxidants. We found that EtOH caused 1) iNOS activation, 2) NO overproduction, 3) increases in oxidative stress and superoxide anion production (superoxide dismutase quenchable fluorescence of dichlorofluorescein), 4) nitration and oxidation of tubulin (immunoblotting), 5) decreased levels of stable polymerized tubulin, and 6) increased levels of disassembled tubulin. EtOH also 7) extensively damaged the MT cytoskeleton and 8) disrupted barrier function. Authentic ONOO(-) or ONOO(-) donors had similar effects. Pretreatment with a selective iNOS inhibitor, L-N(6)-1-iminoethyl-lysine, or with antioxidants (ONOO(-) scavengers urate or L-cysteine; superoxide anion scavenger superoxide dismutase) attenuated damage due to EtOH or to ONOO(-) generators. We conclude that EtOH-induced MT damage and intestinal barrier dysfunction require iNOS activation followed by NO overproduction and ONOO(-) formation. These findings provide a rationale for the development of novel therapeutic agents for alcohol-induced GI disorders that inhibit this mechanism.