The liver is the main organ involved in amino acid metabolism, and it utilizes glucogenic amino acids as substrates for glucose or adenosine triphosphate (ATP), but this process is impaired in clinical and experimental liver diseases. In this study, we administered high doses of amino acids in rats or cultured hepatocytes with experimental models of liver injury to examine whether such supplementation could attenuate liver damage. We found that the addition of alanine reduced enzyme leakage from primary cultured rat hepatocytes treated with D- galactosamine (D-gal), while other amino acids did not. A significant decrease of lactate dehydrogenase (LDH) leakage was observed when cells were cultured with > 6 from mmol/L alanine. Alanine also reduced enzyme leakage from normal hepatocytes that were not treated with D-gal. In D-gal-treated rats, constant infusion of a high dose of alanine significantly reduced the plasma transaminase and total bilirubin levels when compared with infusion of an amino acid mixture. Bolus administration of alanine significantly prevented the elevation of plasma transaminase levels and histological liver damage in CCl4- treated rats, while fructose-1,6 bisphosphate (FDP) had little effect. Alanine might promote the restoration of damaged liver in hepatotoxicant-treated rats, because significant effect was found after the elevation in plasma transaminase levels. Alanine also prevented the decrease of cellular ATP caused by D-gal and appeared to promote ATP production in primary cultured rat hepatocytes. These results indicate that alanine reduces experimental liver damage by a direct effect on hepatocytes.