Activation of the superoxide-generating NADPH oxidase of phagocytes is the result of the assembly of a membrane-localized flavocytochrome (cytochrome b559) with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Activation can be reproduced in an in vitro system in which cytochrome b559-containing membranes are mixed with cytosolic components in the presence of an anionic amphiphile. We proposed that the essential event in activation is the interaction between p67phox and cytochrome b559 and that Rac and p47phox serve as carriers for p67phox to the membrane. When prenylated, Rac can fulfill the carrier function by itself, supporting oxidase activation by p67phox in the absence of p47phox and amphiphile. We now show that a single chimeric protein, consisting of residues 1-212 of p67phox and full-length Rac1 (residues 1-192), prenylated in vitro and exchanged to GTP, becomes a potent oxidase activator in the absence of any other component or stimulus. Oxidase activation by prenylated chimera p67phox (1-212)-Rac1 (1-192) is accompanied by its spontaneous association with membranes. Prenylated chimeras p67phox (1-212)-Rac1 (178-192) and p67phox (1-212)-Rac1 (189-192), containing specific C-terminal regions of Rac1, are inactive; the activity of the first but not of the second chimera can be rescued by supplementation with exogenous nonprenylated Rac1-GTP. An analysis of prenylated p67phox chimeras suggests that the basic requirements for oxidase activation are: (i) a "two signals" membrane-localizing motif present in Rac, comprising the prenyl group and a C-terminal polybasic sequence and (ii) an intrachimeric or extrachimeric protein-protein interaction between p67phox and Rac1, causing a conformational change in the "activation domain" in p67phox.