Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.