Chemokines constitute a protein family that exhibit a variety of biological activities involved in normal and pathological physiological processes. CCL11 (eotaxin), CCL19 (MIP-3beta), CCL22 (MDC), CXCL11 (I-TAC) and CXCL12 (SDF-1alpha) chemokines, modified with the Alexa Fluor 647 fluorescent dye at specific positions along their sequence, were produced by a chemical route and their biological activities were characterized. In a migration assay, fluorescent chemokines were as biologically active as the unmodified forms. All labeled chemokines specifically stained cell lines transfected with the appropriate human chemokine receptors. The specificity of binding was further established by showing that the unlabeled ligands efficiently competed with the labeled chemokines for binding to their respective receptor. A low molecular weight antagonist of CXCR4 prevented binding of labeled CXCL12 to CXCR4 comparably to a neutralizing anti-CXCR4 antibody. Finally, labeled CCL19 was used for the staining of primary cells, illustrating that this reagent can be used for studying CCR7 expression on different cell types. Together, these results demonstrate that fluorescent synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on intact cells, for applications such as cell-based, high throughput screening, and studies of chemokine receptor expression by primary cells.