Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon-gamma is involved in its binding to heparan sulfate. This cluster (Lys125-Arg131) is localized in the C-terminal part of IFN-gamma. Once bound to heparin sulfate, IFN-gamma is protected against protease attack.