Effects on dietary pantethine supplementation on hepatic lipid accumulation and on the activities of lipogenic-related enzymes in the liver were studied in Single Comb White Leghorn laying hens fed isocaloric and isonitrogenous diets containing corn or barley as the carbohydrate source. Addition of 200 ppm pantethine to the corn-soy (CS) basal diet significantly reduced abdominal fat weight, liver triglyceride, as well as total cholesterol and 17 beta-estradiol concentrations in the plasma. Activities of citrate cleavage enzyme (EC 4.1.3.8; CCE) and fatty acid synthetase (FAS) in the liver were significantly reduced when the CS basal diet was supplemented with pantethine, but the activities of nicotinamide adenine dinucleotide phosphate-malate dehydrogenase (EC 1.1.1.40; NADP-MDH) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G6PDH), were not significantly affected. However, liver triglyceride, total cholesterol, and 17 beta-estradiol concentrations in plasma as well as the activities of CCE, FAS, and NADP-MDH in liver were significantly lower in laying hens fed the barley-soy (BS) basal diet than in those fed the CS basal diet. Pantethine supplementation to the BS diet failed to show any significant effect on liver triglyceride content and on the hepatic activities of lipogenic-related enzymes. There were no significant differences in liver weight, rate of egg production, and egg weight among dietary treatments. these results suggest that dietary pantethine is effective in reducing the accumulation of liver and abdominal fat in laying hens fed a CS diet.