The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage λ DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Δ26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 ± 0.6) × 10 5 M-1 in a 50 mM Na+ solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 ± 0.9) × 107 M-1 under the same solution conditions. T7 gp2.5-Δ26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.