A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O₂^-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5′-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (G_ox) that regulates the NADPH oxidase of neutrophils was identified. G_ox was purified and shown to augment the rate of O₂^- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from G_ox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O₂^- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.