Cdc42Hs and Rac1 are members of the Ras superfamily of small molecular weight (p21) GTP binding proteins. Cdc42Hs induces filopodia formation in Swiss 3T3 fibroblasts while Rac1 induces membrane ruffling. Rac1 also activates superoxide production by the components (cytochrome b, p40phox, p67phox, and p47phox) of the neutrophil oxidase. To isolate target proteins involved in these signaling pathways, we have probed proteins from neutrophil cytosol immobilized on nitrocellulose with Cdc42Hs labeled with [gamma-32P]GTP. Cdc42Hs probe detected binding protein(s) of 66-68 kDa in neutrophil cytosol. Rac1 probe also detected the 66-68-kDa proteins, suggesting the possibility that p67phox may be a binding protein for both of these p21 proteins. Indeed, Cdc42Hs and Rac1 were found to bind specifically to purified recombinant p67phox but not the other oxidase components. A 68-kDa Cdc42Hs binding protein was purified from neutrophil cytosol and found to be related to the recently described p65pak kinase from brain. These results suggest that the p68 kinase and p67phox are targets for Cdc42Hs and Rac1 in neutrophils.