A reproducible and quantitative subfractionation of human high density lipoproteins (HDL) by heparin-Sepharose affinity chromatography has been developed. Two elution methods (A and B) were used to subfractionate HDL(2) (d 1.063-1.125 g/ml) or total HDL (d 1.063-1.21 g/ml). Method A separated HDL(2) into three subclasses, each with distinct chemical properties and in vitro metabolic characteristics. The first subclass, referred to as HDL(2)-without E, passed through the affinity column unretarded and represented approximately 85% of the HDL(2) lipoprotein protein. HDL(2)-without E contained the A-I, A-II, and C apoproteins which characterize typical HDL. The second subclass eluted from the column (7-10% of the protein) contained, in addition to the A-I and A-II apoproteins, the E and (E-A-II) apoproteins, and was designated as HDL(2)-with E. The B apoprotein was the major protein component of the third subclass eluted from the column (beta lipoproteins). The beta subclass accounted for approximately 3-8% of the HDL(2) protein and was similar to Lp(a) in composition and size. Method B further subdivided the beta subclass into two fractions (beta(1) and beta(2)) with slightly different electrophoretic mobilities. The various heparin-Sepharose subclasses were further characterized by their ability to compete with (125)I-labeled low density lipoproteins (LDL) for binding to cell surface receptor sites of fibroblasts. By virtue of the presence of the E apoprotein, HDL(2)-with E competed effectively with (125)I-labeled LDL for binding to the cell surface receptors, whereas HDL(2)-without E were ineffective in competing with LDL. The beta subclass possessed binding capability similar to that of LDL. Subfractionation of HDL by heparin-Sepharose affinity column chromatography provides an attractive alternative to methods based solely on ultracentrifugation, in that it subfractionates HDL into subclasses with differing apoprotein contents that impart distinct metabolic characteristics to each class.-Weisgraber, K. H., and R. W. Mahley. Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography.