The RecA protein of Escherichia coli controls the SOS response for DNA damage tolerance and plays a crucial role in recombinational DNA repair. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities, and yet,this process is not understood at the molecular level. An analysis of RecA-DNA interactions was performed using both a mutant RecA protein containing a tryptophan (Trp) reporter and oligodeoxyribonucleotides (ODNs) containing a fluorescent guanine analogue, 6-methylisoxanthopterin (6MI). Experiments using fluorescent ODNs allowed structurally distinct nucleoprotein filaments, formed in the absence and presence of ATPgammaS (a slowly hydrolyzed analogue of ATP), to be differentiated directly. Stopped-flow spectrofluorometry, combined with presteady-state kinetic analyses, revealed unexpected differences in the rates of RecA-ODN and RecA-ATPgammaS-ODN complex assembly. This is the first demonstration that such intrinsically fluorescent synthetic DNAs can be used to characterize definitively the real-time assembly and activation of RecA-ssDNA complexes. Surprisingly, the ssDNA binding event is almost 50-fold slower in the presence of the activating ATPgammaS cofactor. Furthermore, a combination of time-dependent emission changes from 6MI and Trp allowed the first direct chemical test of whether an inactive filament can isomerize to the active state. The results revealed that, unlike the hexameric motor proteins, the inactive RecA filament cannot directly convert to the active state upon ATPgammaS binding. These results have implications for understanding how a coincidence of functions-an ATP-communicated signal-like activity and an ATP-driven motorlike activity-are resolved within a single protein molecule.