Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production [1, 2]. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation [3–10]. Deficiency in PI3Kγ, an upstream regulator of Rac, causes a similar phenotype [11–13]. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac [14], is believed to link GPCRs and PI3Kγ to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1^−/− mouse. P-Rex1^−/− mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1^−/− mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1^−/− neutrophils, but less affected in unprimed or TNFα-primed cells. Recruitment of P-Rex1^−/− neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.