Non-insulin-dependent diabetic (NIDDM) subjects exhibit abnormalities in their plasma lipid and lipoprotein profiles that increase the risk of ischemic heart disease. This study was designed to examine the metabolic behavior of very-low-density (VLDL), intermediate-density (IDL), and low-density (LDL) lipoproteins in NIDDM patients before treatment and after 4 wk of insulin therapy. Basal turnover studies of ¹³¹I-labeled VLDL₁ (svedberg units [Sf] 60–400) and ¹³¹I-labeled VLDL₁ (Sf 20–60) apolipoprotein B (apoB) were conducted in a group of seven NIDDM patients who had been off oral therapy for 1 wk. The subjects exhibited higher than normal transport rates for VLDL₁ and a diminished input of apoB into the VLDL₂ density range. These observations are concordant with the hypothesis that NIDDM patients overproduce VLDL triglyceride but not apoB. VLDL₁ and VLDL₂ were converted to IDL and ultimately to LDL at approximately normal rates, although the delipidation pathway by which apoB-containing particles were processed exhibited different properties from that seen in control subjects. Insulin therapy reduced plasma triglyceride by 38%, and this was associated with a 41% fall in VLDL₁ mass (P < 0.01). VLDL₂ was less affected (19% reduction, P < 0.05), IDL was unchanged, and LDL fell 17% (P < 0.05). Repeat metabolic studies revealed that the major effects of insulin were to reduce VLDL₁-apoB transport (from 811 to 488 mg/day) and increase the direct input of VLDL₂ into the plasma (from 182 to 533 mg/day, P < 0.05). These alterations in VLDL production led to normalization of apoB kinetics in IDL and LDL. The fractional catabolic rate of LDL increased 19% (P < 0.05), whereas direct input into this fraction, which had been high before treatment, was reduced. Postheparin plasma lipoprotein lipase (LPL) and hepatic lipase levels were unaffected by insulin, although the hormone did increase LPL in adipose tissue. This lack of effect on lipase activities correlated well with the observation that the rates of catabolism of apoB in VLDL₁, VLDL₂, and IDL were not significantly affected by insulin therapy.