Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b559 with the cytosolic proteins p47phox and p67phox and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67phox. This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67phox and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2- production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47phox. A chimera consisting of p67phox truncated at residue 212 and fused to a full-length Rac1 [p67phox(1-212)-Rac1 (1-192)] was a potent NADPH oxidase activator. A p67phox(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67phox-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67phox segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47phox. Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67phox(1-212)-Rac1 (1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.