Vav1 is a hemopoietic-specific Rho/Rac guanine nucleotide exchange factor that plays a prominent role in responses to multisubunit immune recognition receptors in lymphoid cells, but its contribution to regulation of neutrophil functions is unknown. Activated Rho family GTPases are critical participants in neutrophil signaling cascades initiated by binding of FMLP and other chemoattractants to their cognate G protein-coupled receptors. Therefore, we investigated whether Vav1 regulates chemoattractant-induced responses in neutrophils. We found that superoxide production elicited by FMLP in Vav1(-/-) murine neutrophils isolated from either bone marrow or from peritoneal exudates was substantially reduced compared with that of wild type. Filamentous actin generation in FMLP-stimulated Vav1(-/-) neutrophils was also markedly reduced, whereas it was normal in response to IL-8 or leukotriene B(4). FMLP induced tyrosine phosphorylation of Vav1, whereas IL-8 or leukotriene B(4) did not, correlating with the requirement for Vav1 in chemoattractant-stimulated filamentous actin generation. Neutrophil motility in vitro and neutrophil mobilization into peripheral blood in vivo elicited by FMLP were both decreased in Vav1(-/-) mice. Hence, this study defines a new role for Vav1 in regulating granulocytic leukocytes as well as linking Vav1 to specific cellular responses downstream of a seven transmembrane domain receptor.