C4b-binding protein (C4BP) is a protein acting as a complement inhibitor and a carrier protein for anticoagulant protein S. Previously, we reported that the in vivo clearance of C4BP involves CD91, and that a CD91-interactive site overlaps the heparin-binding site within C4BP alpha-chains 26. Here, we investigated the C4BP-CD91 interaction in more detail. Binding of C4BP to CD91 was unaffected by protein S, which associates with C4BP beta-chain. Second, mutagenesis of cationic residues within C4BP alpha-chains impaired CD91 binding, reducing the affinity of triple mutant C4BPalpha/R39Q-R64Q-R66Q by 20-fold (Kd= 10 nM versus 214 nM for wild-type and mutant C4BP, respectively). Accordingly, intracellular degradation of this mutant by CD91-expressing cells was reduced to levels of CD91-deficient cells. Moreover, C4BPalpha/R39Q-R64Q-R66Q displayed a 3-fold prolonged survival compared to normal C4BP in in vivo clearance experiments. Since these residues also contribute to heparin binding, we explored the role of heparin-sulfate proteoglycans (HSPG) in the endocytosis of C4BP. The absence of HSPG was associated with a near complete absence of cell binding and intracellular degradation of C4BP. Apparently, the cellular uptake of C4BP depends on both HSPG and CD91, involving interactions with positively charged residues within C4BP alpha-chain.