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Development of specifically labeled chemokines that retain their biological properties should be useful for analyzing their mechanisms of action both under physiological and pathological conditions. Here, we report the chemical synthesis and characterization of RANTES (regulated upon activation normal T cell expressed and secreted) derivatives that were biotinylated at residues 1, 25, 33, 45, or 67. Gel filtration and ultracentrifugation experiments showed that biotinylation at position 45 or 67 decreased the aggregation tendency of the chemokine to a dimeric state. Competition experiments, using a stably transfected CHO-K1 cell line overexpressing human CCR5, a RANTES receptor, indicated that derivatives biotinylated at positions 1, 25, and 67 bound to CCR5 with the same affinity as native RANTES. Flow cytometry analysis showed that RANTES biotinylated at residue 67 (B67-RANTES) bound more efficiently …