Fractalkine, the first member of the CX(3)C chemokine family, induces leukocyte chemotaxis through activation of its high affinity receptor, CX(3)CR1. Like other chemokine receptors, CX(3)CR1 is coupled to a pertussis toxin-sensitive heterotrimeric G(i) protein, which is necessary for rapid rise in the concentration of intracellular calcium. Using a Chinese hamster ovary cell line stably transfected with the CX(3)CR1 receptor, we show that the source of calcium mobilized by fractalkine stimulation is the extracellular pool. Calcium influx is blocked by extracellular calcium chelators, as well as by divalent heavy metals such as Ni(2+), Co(2+), and Cd(2+) without affecting the integrity of intracellular stores. Remarkably, selective phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin and LY294002, abolish the wave extracellular calcium, suggesting that an active PI3K is necessary for this event. The influx of extracellular calcium is in turn required to trigger the activation of the p42/44 mitogen-activated protein/extracellular signal-regulated kinase pathway, but is not necessary for other signals downstream to PI3K, such as phosphorylation of Akt. The potential role of this signaling cascade in fractalkine-mediated chemotaxis is discussed.