Superoxide is the most important armory on the primary defense line of monocytes against invading pathogens, and the identification of new stimuli and the characterization of the regulatory mechanism of superoxide generation are of paramount importance. In this study, we identified 3 novel peptides by screening a synthetic hexapeptide combinatorial library and modification of 1 of the peptides. The isolated peptides that can induce superoxide generation in human monocytes are His-Phe-Tyr-Leu-Pro-Met-CONH(2) (HFYLPM), Met-Phe-Tyr-Leu-Pro-Met-CONH(2) (MFYLPM), and His-Phe-Tyr-Leu-Pro-D-Met-CONH(2) (HFYLPm). All 3 peptides also caused intracellular calcium ([Ca(++)](i)) rise. We tested the specificities of the peptides on cells of different origin by looking at [Ca(++)](i) rise. All 3 peptides acted specifically on leukocytes and not on nonimmune cells. Among leukocytes, HL60 and Jurkat T cells were stimulated specifically by MFYLPM or HFYLPM, respectively. As a physiologic characteristic of the peptides, we observed that all 3 peptides induced chemotactic migration of monocytes. Studying receptor specificity, we concluded that the 3 peptides might act on some shared and some distinct receptor(s) on leukocytes. Studying intracellular signaling set in motion by the peptides revealed that HFYLPM, but not MFYLPM or HFYLPm, induced chemotaxis via phosphatidylinositol-3 kinase and protein kinase C. Because HFYLPM, MFYLPM, and HFYLPm not only exhibit different specificities depending on cell type and status of differentiation but also stimulate cells via distinct receptors and signaling, the 3 novel peptides might be useful tools to study leukocyte activation.