Ephrin-B3 binds HSPGs on HEK-293T, HeLa, and CHO cells, where heparin blocks binding to HEK-293T cells independently of Eph receptors, and a heparin/HS-binding domain in ephrin-B3 was identified outside of the Eph-receptors binding domain. The two positively charged residues, Arg178 and Lys179, in the ephrin-B3's juxtamembrane region is important for heparin/HS binding. Changing the corresponding amino acids in the non-heparin binding ephrin-B1 to positively charged residues gave heparin binding. Ephrin-A3 also binds HS, where Lys176 corresponds to Lys179 in ephrin-B3. Ephrin-B3 binding to lymphocytes and lymphoma cell lines may also depend on other residues near the transmembrane domain, in particular Arg188 which is less affected by heparin, suggesting several mechanisms for ephrin-B3 binding to cells. Functional studies revealed that ephrin-B3 binding to cells induces signaling, influencing both cell rounding and spreading.