Chemoattractants are thought to be the first mediators generated at sites of bacterial infection. We hypothesized that signaling through G protein-coupled chemoattractant receptors may stimulate cytokine production. To test this hypothesis, a human mast cell line (HMC-1) that normally expresses receptors for complement components C3a and C5a at low levels was stably transfected to express physiologic levels of fMLP receptors. We found that fMLP, but not C3a or C5a, induced macrophage inflammatory protein (MIP)-1ss (CCL4) and monocyte chemoattractant protein-1 (CCL2) mRNA and protein. Although fMLP stimulated both sustained Ca(2+) mobilization and phosphorylation of extracellular signal-regulated kinase (ERK), these responses to C3a or C5a were transient. However, transient expression of C3a receptors in HMC-1 cells rendered the cells responsive to C3a for sustained Ca(2+) mobilization and MIP-1ss production. The fMLP-induced chemokine production was blocked by pertussis toxin, PD98059, and cyclosporin A, which respectively inhibit G(i)alpha activation, mitgen-activated protein kinase kinase-mediated ERK phosphorylation, and calcineurin-mediated activation of NFAT. Furthermore, fMLP, but not C5a, stimulated NFAT activation in HMC-1 cells. These data indicate that chemoattractant receptors induce chemokine production in HMC-1 cells with a selectivity that depends on the level of receptor expression, the length of their signaling time, and the synergistic interaction of multiple signaling pathways, including extracellular signal-regulated kinase phosphorylation, sustained Ca(2+) mobilization and NFAT activation.