A disagreement on the mode of histidine binding to copper and the structure of [Cu2+ (HiS)(2)] in solution still exists, Spectroscopic data in solution support a six-coordinate species with N4O2 donor atoms, while X-ray crystallography reveals five-coordinate N3O2 donor atoms. We modified [Cu2+ (HiS)(2)] in solution using diethyl pyrocarbonate (DEPC) and monitored the products spectrophotometrically and by mass spectrometry. Our spectrophotometric study indicates the presence of a free imidazole in the [Cu2+ (HiS)(2)] complex in solution. Mass spectral characterization of a DEPC-modified [Cu2+ (HiS)(2)] complex yielded a peak at 587.8 amu corresponding to three DEPC adducts. Taken together, our data indicate that the [Cu2+ (HiS)(2)] complex in solution exists as a neutral five-coordinate structure with N3O2 donor atoms.