Human plasma high-density lipoprotein-3 (HDL3) has been shown to bind to a variety of cells and tissues. In order to investigate the nature of HDL3-cell association, we studied the interaction of 125I-HDL3 with porcine aortic endothelial cells, rabbit aortic smooth muscle cells, and normal human skin fibroblasts. At 37 degrees C, 125I-HDL3 association with endothelial cells was nonsaturable. Furthermore, 60% protein digestion of HDL3 by trypsin (T-HDL3) actually increased its ability, on a protein weight basis, to associate with endothelial cells and to displace 125I-HDL3 from all three cell types. Synthetic phospholipid-cholesterol discs containing either apo-A-I or apo-A-II were equally effective in displacing 125I-HDL3 from endothelial cells, and phospholipid-cholesterol vesicles containing no protein also displaced 125I-HDL3 from endothelial cells. Neither lipid-free apo-HDL3 nor apo-T-HDL3 was able to competitively displace 125I-HDL3. The above competitive displacement data, when expressed on a protein weight basis, showed differences in the ability of the competitors to displace 125I-HDL3 from cells in the following order of effectiveness: discs greater than T-HDL3 greater than native HDL3. When these data were expressed on a surface lipid weight basis, all three competitors, as well as the lipid vesicles, were approximately normalized to a single competitive displacement curve. Studies on the nature of the cellular mediators of HDL3-cell association revealed that the cell surface sites were resistant to proteolytic treatment. Furthermore, both 125I-HDL3 and 125I-T-HDL3 association with fibroblasts preincubated with varying concentrations of cholesterol increased in parallel with the free cholesterol content of the cells; although cycloheximide blocked this increase in HDL3-cell association, cycloheximide also prevented the increase in cholesterol content of cholesterol-treated cells. We conclude that the association of HDL3 with the cell types studied is not mediated by specific ligand and receptor proteins but rather involves the interaction of cellular surface lipids, possibly cholesterol, with the surface lipids of HDL3.