The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of ATP(gamma)S cofactor using fluorescence anisotropy. RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. Anisotropy signal was shown to measure oligonucleotide binding to RecA and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10^5 M^-1 (37°C, 150 mM NaCl, pH 7.4). The reaction was enthalpy driven and entropy opposed. It depended on salt concentration and was sensitive to the type of monovalent anion suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.