In bacteriophage T4 gene 32 protein (gp32), three sulfur donor atoms, derived from Cys(77), Cys(87), and Cys(90), provide ligands to the Zn(II) ion. Each of the two histidines in the molecule, His(81) and His(64), has been variously proposed to donate the fourth non-thiolate ligand to complete the first coordination shell. In this paper, X-ray and visible absorption spectroscopies have been used to distinguish between these possibilities. The Zn X-ray absorption edge and EXAFS spectra of wild-type and H81A (His(81) --> Ala) gp32s are virtually identical and most consistent with one histidine imidazole ligand with a Zn-N bond distance of 2.06 Angstrom and three S-containing ligands at 2.33 Angstrom. In contrast, as expected for a change in the first coordination shell, the Zn X-ray absorption edge and EXAFS spectra of H64C gp32 are easily distinguished from the wild-type and H81A proteins. In particular the Fourier transform (FT) peaks at R’ approximate to 3 and 4 Angstrom, attributed to the outer shell atoms of histidine imidazole rings, are absent in this mutant. Detailed curve fitting of the EXAFS data, however, do not readily distinguish between S3N1, S(3)imid(1), and S-4 fits. Therefore, Co(II)-substituted H64C gp32 was also characterized. The ligand field transition envelope of the optical absorption spectrum of Co(II) H64C gp32 is significantly red-shifted relative to the wildtype protein, coupled with pronounced changes in the -S- --> Co(II) LMCT region, both consistent with an additional sulfur donor atom to the Co(II) ion. Other substitutions at residue position 64 (H64D and H64L gp32) do not give rise to the same spectral changes. The Co EXAFS of the Co(II) H64C derivative also reveals the apparent absence of outer shell imidazole scattering. These data strongly suggest that His(64) in the wild-type protein (and Cys(64) in H64C gp32) completes the zinc coordination in T4 gene 32 protein.