Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified StSSB by gel filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, StSSB bound to 21-38 nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of StSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 22 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of StSSB for the first tetramer were less than that for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding-mode of StSSB is expected to be noncooperative.