Single bilayer vesicles (d less than 1.02 g/ml) of 3H-glycosphingolipids and [14C]phosphatidylcholine in the molar ratio of 1:7 were prepared by ethanolic injection of the lipid mixture into buffer, concentrated, and incubated with human serum high density lipoprotein-3 (HDL3; d = .14 g/ml) at 37 degrees C. Equilibrium ultracentrifugation of the incubation mixtures on a 0-22% NaBr gradient revealed the presence of three discrete lipid-protein complexes of density 1.03, 1.06, and 1.12 g/ml (Peaks I, II, and III, respectively). Each peak was homogeneous upon reultracentrifugation and the protein and radioactivity eluted as a single peak upon Sepharose CL-6B chromatography. Compositional analysis showed peak I to contain 2.6% protein (apo-A-I peptide) and 4.3% cholesterol, peak II to contain 17.6% protein (apo-A-I peptide) and 6.3% cholesterol, and peak III to have a composition similar to HDL3. Electron microscopy of negatively stained samples confirmed the homogeneity of the peaks and the similarity between peak III and HDL3. Peak II particles were larger than HDL3; peak I particles resembled fused or aggregated vesicles which could be removed by ultracentrifugation; disc-shaped particles were not seen in any of the fractions. Direct incubation of HDL3 or human serum with 3H-glycosphingolipid dispersions did not yield a glycolipid . HDL3 complex as judged by density gradient ultracentrifugation and Sepharose CL-6B chromatography. However, incubation of 3H-glycolipid/phosphatidylcholine vesicles with serum did result in transfer of 3H-glycolipid to the HDL fraction. It was concluded that glycolipids incorporated into a lipid membrane structure can interact with, and become incorporated into, high density lipoprotein.