The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.