This study was undertaken to further investigate the use of the vanadyl ion, V02+, as a spectroscopic probe of active sites in metalloenzymes. The vanadyl derivative of zinc bovine carboxypeptidase A was prepared and found to hydrolyze the ester hippuryl-L-P-phenyllactic acid and the peptide benzoylglycyl-L-phenylalanine. One vanadyl ion per enzyme molecule was sufficient for full activity. Electron paramagnetic resonance (epr) spectra of polycrystalline samples were examined and three types of binding sites, A, B, and C, were found. The A site corresponds to the active site normally occupied by zinc and does not appear unusual in its geometry about the V 0 2 + group. Two water molecules and two imidazole groups, His-69 and His-196, appear to bind in equatorial positions while the axial w e have been investigating the use of the vanadyl ion, V 0 2 + , as a spectroscopic probe of metal binding sites in proteins. In addition to its infrared and visible spectroscopic properties, the vanadyl ion exhibits sharp room temperature and liquid nitrogen electron paramagnetic resonance (epr) spectra which are sensitive to the ligand environment about the V 0 2 + group (Chasteen et al., 1973;Boucher et al.,