In a randomized crossover study, we measured the hepatic secretion rate of very-low-density lipoprotein (VLDL) apolipoprotein B-100 (apoB) in seven patients with well-controlled non-insulin-dependent diabetes mellitus (NIDDM) (HbA1 8.4 +/- 0.4% [mean +/- SE]) on two occasions: during a 13-h hyperinsulinemic (plasma insulin concentration 586 +/- 9.7 pmol/l) euglycemic (plasma glucose concentration 5.2 +/- 0.1 mmol/l) clamp; and during a 13-h saline (control) infusion. After 5 h of the hyperinsulinemic euglycemic clamp (or saline infusion) when a new steady state of apoB turnover was reached, [1-(13)C]leucine was administered by a primed (1 mg/kg), constant 8-h infusion (1 mg.kg-1. h-1). VLDL apoB isotopic enrichment was determined with gas chromatography-mass spectrometry, and a monoexponential model was used to calculate the fractional secretion rate of VLDL apoB. VLDL apoB secretion rate was significantly reduced during the hyperinsulinemic euglycemic clamp compared with the saline study (12.2 +/- 3.6 vs. 24.5 +/- 7.1 mg.kg-1.day-1, P = 0.001), but there was no change in the fractional catabolic rate of VLDL apoB. Concomitantly, plasma concentrations of nonesterified fatty acids (NEFAs), glycerol, and triglycerides (TGs) were significantly lower during the hyperinsulinemic euglycemic clamp compared with the saline study (NEFAs, P < 0.001; glycerol, P = 0.005; TGs P = 0.004). We conclude that acute hyperinsulinemia decreases the hepatic secretion rate of VLDL apoB in NIDDM, probably in part due to reduction in the delivery of NEFA and glycerol substrate to the liver.