Several serpin-enzyme complexes bind to a receptor on hepatocytes that mediates their endocytosis and lysosomal degradation. Joslin et al. (Joslin, G., Fallon, R. J., Bullock, J., Adams, S. P., and Perlmutter, D. H. (1991) J. Biol. Chem. 266, 11282-11288) proposed that a sequence near the C-terminal end of the serpin (e.g. FV-FLM in α1-antitrypsin) binds to the serpin- enzyme complex receptor (SEC receptor). In experiments with synthetic peptides, they found that substitution of alanine at the fourth or fifth position in this sequence reduced the affinity of peptide binding to Hep G2 cells. To test the hypothesis that the corresponding sequence in heparin cofactor II (HCII), FLFLI (residues 456-460), mediates binding and uptake of the thrombin-HCII complex by Hep G2 cells, we constructed five recombinant HCII variants, F456A, L457A, F458A, L459A, and I460A. At 4 °C, the 125I- thrombin-HCII(native) complex bound reversibly to 0.6-2.6 x 105 sites per Hep G2 cell with a K(d) of 19-32 nM. Binding was inhibited by excess unlabeled thrombin-HCII(native), thrombin-antithrombin, or elastase-α1- antitrypsin, but not by free HCII or thrombin, which is consistent with the reported specificity of the SEC receptor. However, complexes of thrombin with each of the HCII variants inhibited binding as effectively as the complex with native HCII. Competitive binding experiments with various concentrations of unlabeled thrombin-HCII(native) or thrombin-HCII(I460A) indicated that these complexes bind to Hep G2 cells with equal affinity. At 37 °C, complexes of 125I-thrombin with each of the five HCII variants were internalized and degraded at the same rate as the complex with native HCII. Our data suggest that the pentapeptide FLFLI in HCII is not involved in binding, internalization, and degradation of thrombin-HCII complexes by Hep G2 cells.