Hypertriglyceridemia is associated with increased risk of cardiovascular disease. Until recently, the importance of hepatic de novo lipogenesis (DNL) in contributing to hypertriglyceridemia was difficult to assess because of methodologic limitations. We evaluated the extent of the contribution by DNL to different conditions associated with hypertriglyceridemia. After 5 d of an isoenergetic high-fat, low-carbohydrate diet, fasting DNL was measured in normoinsulinemic (or= 115 pmol/L) obese (n = 8) subjects. Fasting DNL was measured after a low-fat, high-carbohydrate diet in normoinsulinemic lean (n = 5) and hyperinsulinemic obese (n = 5) subjects. Mass isotopomer distribution analysis was used to measure the fraction of newly synthesized fatty acids in VLDL-triacylglycerol. With the high-fat, low-carbohydrate diet, hyperinsulinemic obese subjects had a 3.7-5.3-fold higher fractional DNL (8.5 +/- 0.7%) than did normoinsulinemic lean (1.6 +/- 0.5%) or obese (2.3 +/- 0.3%) subjects. With the low-fat, high-carbohydrate diet, normoinsulinemic lean and hyperinsulinemic obese subjects had similarly high fractional DNL (13 +/- 5.1% and 12.8 +/- 1.4%, respectively). Compared with baseline, consumption of the high-fat, low-carbohydrate diet did not affect triacylglycerol concentrations. However, after the low-fat, high-carbohydrate diet, triacylglycerols increased significantly and DNL was 5-6-fold higher than in normoinsulinemic subjects consuming a high-fat diet. The increase in triacylglycerol after the low-fat, high-carbohydrate diet was correlated with fractional DNL (P < 0.01), indicating that subjects with high DNL had the greatest increase in triacylglycerols. These results support the concept that both hyperinsulinemia and a low-fat diet increase DNL, and that DNL contributes to hypertriglyceridemia.