Leukocytes are key cellular components of innate immunity. These phagocytic cells respond to bacteria at sites of infection through chemotactic sensing and directed motility regulated by Rho GTPases. The development of sensitive probes of Rho GTPase dynamics has provided insights into the temporal and spatial aspects of GTPase regulation during chemotaxis and subsequent microbial phagocytosis. The resulting destruction of ingested bacteria by means of reactive oxygen species (ROS) depends on a Rac-regulated ‘molecular switch' that is modulated by antagonistic crosstalk involving Cdc42. Recent studies of leukocytes derived from Rac1- and Rac2-knockout mice have shown that these highly homologous GTPases have unique biological roles. An understanding of the biochemical basis for such distinct activities should provide novel insights into the molecular details of Rho GTPase function and regulation in innate immunity.