The mechanism by which alcohol injures the pancreas remains unknown. Recent investigations suggest a role for fatty acid ethyl ester (FAEE), a nonoxidative metabolite of ethanol, in the pathogenesis of alcohol pancreatitis. In this study, we characterized ethanol-induced injury in rats and evaluated the contribution of oxidative and nonoxidative ethanol metabolites in this form of acute pancreatitis. Pancreatic injury in rats was assessed by edema, intrapancreatic trypsinogen activation, and microscopy after infusing ethanol with or without inhibitors of oxidative ethanol metabolism. Plasma and tissue levels of FAEE and ethanol were measured and correlated with pancreatic injury. Ethanol infusion generated plasma and tissue FAEE and, in a dose-dependent fashion, induced a pancreas-specific injury consisting of edema, trypsinogen activation, and formation of vacuoles in the pancreatic acini. Inhibition of the oxidation of ethanol significantly increased both FAEE concentration in plasma and pancreas and worsened the pancreatitis-like injury. This study provides direct evidence that ethanol, through its nonoxidative metabolic pathway, can produce pancreas-specific toxicity in vivo and suggests that FAEE are responsible for the development of early pancreatic cell damage in acute alcohol-induced pancreatitis.