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The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, ⁴⁴RKNR⁴⁷ and⁵⁵KKWVR⁵⁹. The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the⁴⁴AANA⁴⁷ mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the⁵⁵AAWVA⁵⁹ mutant retained full binding capacity. Mutation of the ⁴⁴RKNR⁴⁷ site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity …