Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 13Cd resonance, δ 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a δ and a line width the same as those observed for the wild-type protein. Thus, the S−ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligandprotons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA. The visible absorption spectra of the Co(II) derivative of g32P and its Ser166 mutant suggest an approximately tetrahedral ligand field around the Co(II) ion in g32P. 35C1 NMR shows that the Zn(II) site is not accessible to anions from solution, which suggests that a fourth ligand from the protein completes the tetrahedral complex. The best candidate is an imidazole nitrogen from His81. A simple molecular modeling procedure shows that a series of peptide turns compatible with peptide backbone angles found in proteins can accommodate the formation of a tetrahedral Zn(II) complex with Cys77, His81, Cys87, and Cys90 as ligands.