Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac [1–4]. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils [5–8], but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions [9–13]. Here, we characterized P-Rex1, a Gβγ and PIP₃-regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants [14], for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished Rac2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for Rac2 may partially result from the apparent higher affinity of P-Rex1 for Rac2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with Rac2_S17N than Rac1_S17N. In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.