Determination of quantitative de novo fat synthesis rates is important in our understanding of the role of nutritional factors in macronutient utilization and obesity development. To address these questions, we developed and applied a method for measuring lipogenesis using the uptake of deuterium from heavy water into triglyceride fatty acids (TGFA) in humans. Our model examined the plateau of deuterium enrichment in plasma very light density lipoprotein (VLDL) TGFA relative to the maximum as indicative of the fraction of VLDL-TGFA synthesized. Plateau enrichment was observed after 12 h of prime-constant oral deuterium oxide administration. Our initial studies applied this model in healthy humans consuming typical diets to demonstrate rates of TGFA synthesis amounting to 2 g.day-1. In subsequent experiments it was shown that this level of synthesis was not affected by the frequency with which daily meals were consumed. Furthermore, in a clinical application, our results using this approach demonstrated that compared with normolipidemic controls, individuals with type III hyperlipoproteinemia displayed elevated TGFA but normal cholesterol synthesis rates. In summary, compared with other available methods for determination of lipogenesis, deuterium uptake is safe, direct, and inexpensive. Results suggest that under most dietary circumstances, human de novo lipogenesis is minor, thus dietary fat and carbohydrate balances remain largely independent.