RecA protein from Escherichia coli catalyzes DNA strand exchange during homologous recombination in a reaction that requires nucleoside triphosphate cofactor. In the first step of this reaction RecA protein polymerizes on single-stranded DNA to form a filament with a stoichiometry of 3 nucleotides/RecA monomer called the presynaptic complex. We have used fluorescence anisotropy of a fluorescein labeled oligonucleotide to investigate presynaptic complex formation. RecA-ATP(gamma)S bound to oligonucleotide by a two-step process. Kinetic studies revealed an intermediate in the polymerization reaction which had greater mobility than the final product filament. The intermediate was transformed into final product by a process which was independent of filament concentration and temperature, k = 0.3 +/- 0.1 min-1. This process had the same rate as that reported for a step in the isomerization of presynaptic complex by ATP(gamma)S (Paulus and Bryant (1997) Biochemistry 36, 7832). Judging from anisotropy measurements, the intermediate had hydrodynamic properties similar to a mixed filament containing RecA monomers with and without ATP(gamma)S. These results show that the presynaptic complex can assume conformations with different segmental mobilities which could play a role in homologous recombination.