It has been suggested that pyridoxine deficiency may potentiate ethanol-induced liver injury. Our purpose was to clarify the effect of pyridoxine deficiency on ethanol-associated liver injury by comparing liver histology, serum liver enzymes, and the viability of cultured hepatocytes from pyridoxine-deficient and pyridoxine-sufficient rats that had been chronically fed ethanol-enriched diets. Our data fail to substantiate that pyridoxine-deficient animals are more susceptible to the hepatotoxic effects of ethanol than pair-fed pyridoxine-sufficient controls. Furthermore, the addition of pyridoxine to hepatocyte cultures fails to prevent in vitro cytotoxicity of added ethanol. Pyridoxine deficiency may augment ethanol-induced enhancement of hepatic urea synthesis. These data suggest that pyridoxine deficiency may contribute to the abnormal plasma amino acid profiles and nitrogen balance of chronic alcoholics, but that it does not potentiate ethanol-induced liver injury.