Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in Gαi protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCβ is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3), and demonstrate that loss of PLCβ2 and PLCβ3 significantly impaired T cell migration. Because second messengers generated by PLCβ lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCβ-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1α was undetectable in PLCβ2β3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCβ play a critical role in T lymphocyte chemotaxis. Copyright © 2007 by The American Association of Immunologists, Inc.