Bacteriophage T4-coded gene 32-protein is an essential component of the T4 replication and recombination systems. Alberts and co-workers (Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 289-305) have shown that the major physiological activity of the protein involves preferential and cooperative binding to single-stranded DNA. In this paper, the physiochemical parameters characterizing this melting protein system are quantitatively determined. Boundary sedimentation velocity experiments are used to measure the interaction of gene 32-protein with native DNA. The binding is shown to be non-cooperative and involves an overlapping site size (nh) of approximately 10 nucleotide residues (or approximately 5 nucleotide pairs). In analogy with the ribonuclease results (Jensen, D.E., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7198-7214), the logarithm of the association constant (Kh) is found to be linerarly related to log [Na+]. The binding of gene 32-protein to denatured (single-stranded) DNA involves appreciable distortion of the polynucleotide backbone from the unliganded conformation; binding totally unstacks the bases of both ribose- and deoxyribose-containing polynucleotides at 10 degrees, and results in a hyperchromic change exceeding that which can be induced by heating. This hyperchromism induced in poly(dA) on binding gene 32-protein under low salt (tight binding) conditions is used to determine a value of nc (the single-stranded DNA site size) of approximately 6.7 nucldotide residues per protein. In addition, gene 32-protein binding to single-stranded polynucleotide induces an unusual circular dichroic spectrum characterized principally by a marked decrease in the magnitude of the positive CD band centered at approximately 265 nm. This spectral change is attributed to significant uncoupling of the transition moments of the vicinal bases of the single-stranded polynucleotide on gene 32-protein binding, in accord with the ultraviolet hyperchromism observed. Binding of gene 32-protein to double helical DNA has virtually no effect on the spectral properties of this conformation...