Acute pancreatitis is a serious and often lethal inflammatory disease. Its causes are diverse and incompletely understood; however, gallstones and alcohol abuse are the principal triggers. Oxidative stress has been proposed as a determinant of acute pancreatitis (AP) severity, and has been the subject of recent clinical trials. The major AP precipitants, alcohol, alcohol metabolites and bile salts, were investigated for their potential role in the production of reactive oxygen species (ROS), and their effects upon cell fate. Application of the bile salt taurolithocholic acid sulphate (TLC-S) to isolated human and murine pancreatic acinar cells generated significant Ca2+-dependent mitochondrial ROS which were inhibited with the antioxidant N-acetyl-L-cysteine (NAC), and promoted with dimethoxy-2-methylnaphthalene (DMN), an inhibitor of the antioxidant enzyme NAD(P)H quinone oxidoreductase (NQO1). Elevations of ROS mediated by bile salts were crucial in the determination of cell fate, producing apoptosis rather than necrosis. In contrast, ethanol and its metabolites, both oxidative (acetaldehyde) and non-oxidative (fatty acid ethyl esters: FAEEs), were shown to produce no significant ROS in similar circumstances. Assessment of ethanol and its metabolites revealed that ethanol and acetaldehyde showed little effect on cell fate. Low concentrations of ethanol with fatty acid, however, induced toxic elevations of [Ca2+]C, mitochondrial dysfunction and necrosis when oxidative metabolism was compromised. This effect was reversed by inhibition of FAEE synthase, suggesting important deleterious actions of non-oxidative alcohol metabolism in the pancreas.