We have probed an epitope sequence (His18-Pro19-Lys20-Phe21) in interleukin-8 (IL-8) by site-directed mutagenesis. This work shows that single and double Ala substitutions of His18 and Phe21 in IL-8 reduced up to 77-fold the binding affinity to IL-8 receptor subtypes A (CXCR1) and B (CXCR2) and to the Duffy antigen. These Ala mutants triggered neutrophil degranulation and induced calcium responses mediated by CXCR1 and CXCR2. Single Asp or Ser substitutions, H18D, F21D, F21S, and double substitutions, H18A/F21D, H18A/F21S, and H18D/F21D, reduced up to 431-fold the binding affinity to CXCR1, CXCR2, and the Duffy antigen. Interestingly, double mutants with charged residue substitutions failed to trigger degranulation or to induce wild-type calcium responses mediated by CXCR1. Except for the H18A and F21A mutants, all other IL-8 mutants failed to induce superoxide production in neutrophils. This study demonstrates that IL-8 recognizes and activates CXCR1, CXCR2, and the Duffy antigen by distinct mechanisms.