We have investigated hepatic de novo lipogenesis and the ratio of apoB-48/apoB-100 during chronic carbohydrate supplementation with or without administration of exogenous insulin in rats. Two groups received chronic (2 weeks) carbohydrate supplementation either as 10% glucose or 10% fructose (wt/v) in their drinking water. Two other groups received exogenous insulin chronically, in addition to the monosaccharides above. The insulin was given for 2 weeks as daily human ultralente insulin injections in increasing doses up to 6 units per day. A fifth group of rats (normal control) received only chow and water. The fractional synthetic rate (FSR), the fraction of very low density lipoprotein triglyceride (VLDL-TG) palmitate that was newly made during an 8-h infusion with sodium [1-13C]acetate, was evaluated. The glucose and fructose groups had a 4-fold (0.60%/h) and 7.5-fold (1.13%/h) increase in FSR from baseline, respectively, compared to chow-fed controls (0.15%/h). Chronic exogenous insulin administration resulted in a 11.5 (1.73%/h) and 11.0 (1.65%/h)-fold increase over baseline in the synthesis of newly made VLDL-TG palmitate in the glucose and fructose groups, respectively. The ratio of apoB-48/apoB-100, i.e. apoB-48 enrichment, in VLDL was positively correlated with insulin levels (r = 0.41, P < 0.01) and with FSR (r = 0.39, P < 0.01). The present study shows that carbohydrate supplementation significantly increases the FSR of newly made VLDL-TG palmitate and that this increase is further augmented by chronic hyperinsulinemia.