Esterified cholesterol transfer (ECT) from high density lipoproteins (HDL) to very low (VLDL) and low density lipoproteins (LDL) may be abnormal in situations at high risk for atherosclerosis. It has been shown to increase in insulin-dependent diabetes and to decrease in non-insulin-dependent diabetes (NIDD). Since the net transfer of esterified cholesterol (EC) results from a bidirectional exchange between HDL and VLDL/LDL, we developed a transfer assay specifically designed to measure the unidirectional transfer of EC from HDL to lipid emulsions according to first-order kinetics. Our results show that in NIDD the rate constant of HDL-dependent ECT is decreased by 30% by comparison with control subjects. Analysis of HDL composition revealed that, in both groups, HDL-dependent ECT was positively correlated with the free cholesterol/phospholipid ratio (r = 0.94; P < 0.001) and negatively correlated with the triglyceride/EC ratio (r = -0.85; P < 0.001). It is concluded that, besides the known defect of acceptor lipoproteins, the abnormality of ECT in NIDD is also caused by a decreased ability of HDL to act as an EC donor, presumably because of a change in composition. In addition, our work shows that the amount of EC lost by HDL during the reaction transfer is counterbalanced by a reciprocal equimolar transfer of triglycerides.