High density lipoprotein (HDL) subfractions (2b, 2a, 3a, 3b, and 3c) separated by gradient gel electrophoresis (GGE) and defined by Gaussian summation analysis, and the compositions of HDL2 and HDL3, separated by preparative ultracentrifugation, were studied in four groups of men with or without non-insulin-dependent diabetes mellitus (NIDDM) and coronary artery disease (CAD): group 1 (DM+CAD+, n = 50); group 2 (DM-CAD+, n = 50); group 3 (DM+CAD-, n = 50); and group 4 (DM-CAD-, n = 31). HDL GGE subfraction distributions, available in 125 subjects, were not significantly different among the groups. In contrast, dividing the whole study population into quartiles of serum triglyceride (TG) concentration showed that high TG levels were significantly associated with low HDL2b and high HDL3b concentrations. In a multivariate linear regression model, postheparin plasma hepatic lipase (HL) activity, and fasting serum insulin and TG concentrations were all associated independently and inversely with low HDL2b, but lipoprotein lipase or cholesteryl ester transfer protein activities were not correlated with HDL2b concentrations. Group 1 tended to have the smallest mean particle sizes in the HDL subfractions, significantly (P < 0.03, CAD vs. non-CAD) for HDL2b and for HDL2a. These differences were independent of TG, insulin and HL, but lost their significance when adjusted for beta-blocker therapy. Both HDL2 and HDL3 particles in group 1 were significantly depleted of unesterified cholesterol, and their HDL2 was TG-enriched (P = 0.053). A high HL activity, hyperinsulinemia and hypertriglyceridemia are independently associated with low levels of HDL2b and generally small HDL particle size. HDL particles in subjects with NIDDM and CAD are small-sized and have a low free cholesterol content. Both these characteristics may be markers of impaired reverse cholesterol transport.