In neutrophils, an intracellular calcium ion plays an important role in regulating cellular functions induced by chemoattractants. In this study, we have investigated the role of intracellular calcium and its effecter molecule, calmodulin, in superoxide anion production and chemotaxis of rat neutrophils. N-formyl-methionylleucyl-phenylalanine (FMLP), one of the chemoattractants, is known to activate phosphlipase C, phosphatidyl inositol 3-kinase, and Akt via activation of its G protein-coupled membrane receptors. Inositol trisphosphate produced by both enzymes increases a release of calcium ion from the store. By inhibition of calcium release, the production of superoxide anion in neutrophils was abrogated, whereas the chemotaxis was unaffected. In contrast, both the superoxide anion production and the chemotaxis of neutrophils were suppressed by inhibition of influx of extracellular calcium ion. Inhibition of calmodulin using a calmodulin antagonist, W-7, attenuated FMLP-induced superoxide anion production and chemotaxis of neutrophils. W-7 also inhibited FMLP-induced Akt phosphorylation. Taken together, our data indicate that a critical role of calcium ion and calmodulin in superoxide anion production and chemotaxis of neutrophils, and suggested that FMLP-induced Akt activation may be meditated by calcium-calmodulin pathway in rat neutrophils.