The functional role of Zn(II) binding by T4 gene 32 protein (gp32), a single-stranded DNA-binding protein, has been investigated by assessing the capacity of a well-characterized metal-free gp32 derivative to function in vitro as an accessory protein of T4 uvsX-catalyzed homologous pairing. Metal-free gp32 was prepared upon reaction of cysteine thiolates with methylmethanethiol-sulfonate to form the mixed disulfide Cys-SSCH3 or S-methylated species. Far and near ultraviolet circular dichroism spectroscopy suggest a moderate but easily detected change in the far UV region, accompanied by only a minor alteration in the near UV region, relative to the Zn(II)-containing protein. Restoration of the wild-type spectral features is accomplished upon the addition of 2 mM dithiothreitol and excess Zn(II) but not dithiothreitol alone. Unlike wild-type gp32, apo S-methylated gp32 shows weak binding to the recombination substrate, single-stranded M13mp19, and fails to stimulate homologous pairing with a linear M13mp19 duplex substrate by uvsX protein. Complete reactivation of the apo S-methylated protein as a recombination-accessory protein is achievable in situ in the presence of reducing agent and sufficient exogenous Zn(II), but not one or the other alone. Analogous results are obtained with S-methylated C166S (Cys166-->Ser) gp32, revealing that only the metal-liganding cysteines participate in the reconstitution. These findings suggest that formation of the Zn(II) chelate is directly linked to single-stranded DNA binding and functional efficacy of gp32 in DNA metabolism.