Newly synthesized triglyceride (TG) may exit the liver immediately as VLDL-TG or be stored and secreted after a delay. We quantified the contributions from plasma NEFA, diet, and de novo lipogenesis (DNL) to VLDL-TG via immediate and delayed pathways in five lean, normolipidemic subjects; six obese, hypertriglyceridemic (HPTG) nondiabetics; and six obese, HPTG diabetics. Intravenous 2H31palmitate and 1-13C1 acetate and oral 2H35stearate were administered for 30 h preceding an overnight fast. 1,2,3,4-13C4palmitate was infused during the subsequent 12 h fast. Contributions from plasma NEFA via the immediate pathway were 64 ± 15, 33 ± 6, and 58 ± 2% in control, HPTG, and diabetic HPTG, respectively. Delayed pool fractional contributions were as follows: dietary FA, 2.0 ± 0.9, 2.5 ± 1, and 12 ± 2%; DNL, 3 ± 0.3, 14 ± 3, and 13 ± 4%; delayed NEFA, 15 ± 4, 20 ± 4, and 30 ± 3%. VLDL-TG production rates and absolute input rates from the delayed pool were significantly higher in HPTG and diabetic HPTG than in controls. In conclusion, we provide direct kinetic evidence for a hepatic TG storage pool in humans and document its metabolic sources. The turnover time and sources of this pool differ in diabetic HPTG and nondiabetic HPTG, with potential therapeutic implications.