Chemoattractants induce a variety of phagocytic functions including transendothelial migration, degranulation, and the generation of superoxide anions. We report here that the prototypic chemotactic peptide fMet-Leu-Phe (fMLF) stimulates the activation of nuclear factor-κB (NF-κB), a transcription factor that is central to the regulation of proinflammatory immediate-early gene expression. In freshly prepared peripheral blood mononuclear cells, fMLF (1-100 nM) induced a κB binding activity that was receptor-dependent and involved the p50 and p65 subunits of the NF-κB/Rel family of proteins. The activation of NF-κB by fMLF appeared to be cell- specific and different from the activation of NF-κB by tumor necrosis factor-α (TNFα). Neutrophil preparations that responded to fMLF, TNFα, and lipopolysaccharides with interleukin-8 secretion did not show NF-κB activation, whereas N-formyl peptide receptor (FPR)-transfected HL-60 cells were responsive to TNFα but not fMLF for NF-κB activation. Differentiation of FPR-transfected HL-60 cells with dimethyl sulfoxide for 3-5 days conferred the capability of the cells to activate NF-κB in response to fMLF without a significant increase in the amount of FPR. These results identify NF-κB as a transcription factor that can be activated by the prototypic chemotactic peptide and demonstrate that this function is both highly regulated and dependent on signaling components specifically expressed during myeloid differentiation.