The contribution of circulating free fatty acids (FFA) to plasma triglyceride fatty acid (TGFA) formation has been studied. Isotopically labeled palmitic acid was infused at a constant rate for many hours to ensure that the TGFA in very low density lipoproteins (VLDL) had fully equilibrated with all precursors. Once a constant VLDL TGFA specific activity had been achieved the ratio of the specific activity in VLDL TGFA to that in plasma FFA provided a measure of the contribution of plasma FFA to TGFA. In seven healthy volunteers and in two subjects with endogenous hypertriglyceridemia who were fasted overnight following normal diets, this ratio (expressed as a percentage) was 80–100%. In five subjects with alcoholic fatty liver it was less than 50%. In two of three subjects on clofibrate therapy it was less than 40%; 8 wk after cessation of clofibrate the ratio had returned to above 80%. Three of the normal subjects were restudied during the course of prolonged glucose consumption and then the ratio was only 30–60%. Simultaneous infusion of ¹⁴C-U-glucose in two of these subjects revealed that, in the absence of ¹⁴C-label appearing in plasma FFA, 80% of the ¹⁴C label that appeared in plasma triglyceride was in the fatty acid moiety (TGFA). This incorporation of ¹⁴C-glucose label into TGFA persisted for up to 12 hr after the last meal in subjects who had been eating high carbohydrate diets. In normally fed subjects studied after an overnight fast, ¹⁴C-glucose label appeared only in the glycerol moiety of plasma triglyceride. We conclude that in healthy subjects eating normal diets, plasma TGFA newly formed in the postabsorptive state is derived predominantly from plasma FFA. During glucose consumption, however, hepatic lipogenesis is significant. Stored hepatic fatty acids appeared to be a source of plasma TGFA in the subjects with alcoholic fatty liver and in those treated with clofibrate.