Classical chemoattractant receptors are of fundamental importance to immune responses. The two major roles of such receptors are the modulation of chemotaxis and the generation of reactive oxygen species. The formyl peptide receptor-like 1 (FPRL1) can be stimulated by two different ligands, Trp-Lys-Tyr-Met-Val-Met-CONH2 (WKYMVM) and lipoxin A4 (LXA4). Although leukocyte chemotaxis mediated by activated FPRL1 has been reported, the role of FPRL1 in superoxide generation remains to be studied. In this study, we examined the effect of WKYMVM or LXA4 on chemotactic migration and superoxide generation in human neutrophils. WKYMVM and LXA4 stimulated neutrophil chemotaxis via tyrosine phosphorylation events. In terms of reactive oxygen species generation, WKYMVM but not LXA4 stimulated superoxide generation in neutrophils. To understand this difference on superoxide generation via the same receptor, FPRL1, we compared the signaling pathways downstream of FPRL1 by the two different ligands. At first, we confirmed that both WKYMVM and LXA4 caused intracellular calcium ([Ca2+]i) increase in a pertussis toxin-sensitive manner and that these ligands competitively inhibited each other with respect to [Ca2+]i increase in neutrophils. This result suggests that WKYMVM and LXA4 share the same receptor, FPRL1. By investigating cellular signaling by WKYMVM and LXA4, we found that WKYMVM but not LXA4 induced extracellular signal-regulated protein kinases (ERKs), c-Jun NH2-terminal kinase, and phospholipase A2 (PLA2) activation. We also found that ERK-mediated cytosolic PLA2 activity is essential for superoxide generation. These results indicate that the activation of FPRL1 by the two different ligands can induce differential cellular signaling and unique functional consequences in human neutrophils.