Sources of fatty acids flowing to the liver may be used for triacylglycerol (TAG) synthesis. Our objective was to quantify contributions of nonesterified fatty acids (NEFAs), de novo lipogenesis, and dietary fatty acids to VLDL-TAG in the fed state after meal feeding in healthy subjects (n = 6). The effect of substrate delivery rate was also determined by comparison with data obtained under a continuous-feeding regimen. A liquid diet was administered by mouth or via feeding tube. Contributions of NEFAs, de novo lipogenesis, and dietary fatty acids to VLDL-TAG were quantified using stable isotopes and gas chromatography-mass spectrometry. Contribution of NEFAs to VLDL-TAG was similar under meal feeding and continuous feeding, although insulin area under the curve (AUC) was greater under meal feeding (1,597 +/- 455 vs. 471 +/- 484 pmol . h . l(-1), P < 0.004). Lipogenesis achieved a higher AUC with meal feeding versus continuous feeding (88.7 +/- 84.4 vs. 1.9 +/- 19.3 mumol . h . l(-1), P = 0.03) supporting greater stimulation of de novo lipogenesis from increased glucose delivery rate. The contribution of dietary fatty acids to VLDL-TAG was also greater with meal feeding. These data demonstrate for the first time in humans the well-coordinated use of fatty acids by the liver during the transition from fasted to fed states and highlight the dominant role of NEFAs for VLDL-TAG synthesis in both states.