Human chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. A previous study demonstrated that CKLF1 is a functional ligand for human CC chemokine receptor 4 (CCR4). The N-terminal amino acid sequencing of secreted CKLF1 protein showed that it contains at least two peptides, CKLF1-C27 and CKLF1-C19, which have functional activation via CCR4. To quantitatively evaluate the interaction of CKLF1-C27 or CKLF1-C19 with CCR4, the N-terminal extracellular tail of CCR4 (ML40), CKLF1-C27 and CKLF1-C19 were chemically synthesized and analyzed by capillary zone electrophoresis. Both qualitative and quantitative characterizations of the peptide-peptide binding were determined. We used the macrophage-derived chemokine (MDC) as the positive control and its binding constant was (4.99 ± 0.86) × 10 M. The binding constant of the interactions between CKLF1-C27/CKLF1-C19 and ML40 was calculated as (2.96 ± 0.59) × 10 M and (1.39 ± 0.38) × 10 M by the Scatchard analysis. This result proved that CKLF1-C27 had a greater potent affinity with ML40 than CKLF1-C19 because of the excess eight amino acids. To understand the molecular basis for the interaction, a mutagenesis study of CKLF1-C19 was undertaken. CKLF1-C19pm and CKLF1-C19km were synthesized and their interactions with ML40 were analyzed by CZE. We found that the substitution of Lys or Pro to Ala within the residues of CKLF1-C19 strongly abolished or decreased its interaction with ML40, suggesting that the Lys residues of CKLF1-C19 play the important role for the interaction and the Pro residues of CKLF1-C19 affect its affinity. All the experimental results show that the reported method by CZE for the determination of the interactions of CKLF1 peptides and the N-terminal extracellular tail of CCR4 is powerful. © 2009 Vieweg+Teubner | GWV Fachverlage GmbH.