The LDL receptor-independent binding of human apolipoprotein E isoforms and rare apoE mutations were studied on LDL receptor-deficient human fibroblasts using chemical cross-linking and cell binding studies. The cross-linking experiments demonstrated that all apoE variants bind to the low density lipoprotein receptor-related protein, a potential receptor for remnant lipoproteins. In cell binding studies, the effect of the apoE variants on binding of beta-VLDL was investigated. Addition of normal apoE-3 to the binding assay resulted in a 12-fold increase of beta-VLDL particle binding, whereas this effect was reduced in the clinically defective variants: apoE-2, (Arg158–>Cys), 24.4% of apoE-3; apoE-1, (Gly127–>Asp, Arg158–>Cys), 49.2%of apoE-3; apoE-1(Lys146–>Glu), 18.2%of apoE-3. Heparin binding studies with the same variants showed a parallel reduction in proteoglycan binding (apoE-2(158), 58.2%of apoE-3; apoE-1(127,158), 37.9%; apoE-1(146), 20.6%). We conclude that LDL receptor-independent mechanisms contribute to remnant clearance. The functionally dominant mutation apoE-1(146) was most defective in heparin binding studies in vitro. In cell binding studies, apoE-1(146) did mediate lipoprotein binding only 18%compared to apoE-3. This indicates the important role of the apoE interaction with proteoglycans in vivo and could explain the development of type III hyperlipoproteinemia in patients with such apoE variants.