So far chemokine antagonists have been identified by modification of the NH2-terminus of known chemokines or by screening large number of compounds in functional assays. Here we used phage display peptide libraries to identify hexapeptides that antagonize the interaction between eotaxin and its receptor CCR3. The peptide sequence CPWYFWPC was recovered by panning phage libraries on CCR3-transfected murine pre-B cells after elution with eotaxin. The synthetic, structurally constrained peptide effectively competed 125I-eotaxin binding to CCR3 (IC(50) = 20 microM). Furthermore, it had weak agonistic effects on Ca(2+) mobilization in CCR3 transfectants that underwent heterologous desensitization by subsequent exposure to eotaxin. The peptide inhibited chemotaxis of CCR3 transfectants induced by a broad panel of CCR3 ligands. Specificity was tested with the CXCR1, CXCR2, CXCR3 and CCR5 receptors. In experiments aimed at characterization of residues necessary for eotaxin binding, we affinity purified the linear eotaxin-binding peptide VTPRQR, and showed that the peptide displaced the binding of radiolabeled eotaxin to CCR3 (IC(50) = 300 microM) ina dose-dependent manner, inhibited eotaxin induced increases in intracellular Ca(2+), and migration of CCR3-transfected cells. Specificity was affirmed using other CCR3 ligands. This is the first de novo identification of chemokine antagonists by direct screening on target proteins.