The structures of the dinuclear Ni(II) active sites of urease from jack bean and Klebsiella aerogenes are compared with and without the addition of the inhibitor 2-mercaptoethanol (2-ME). No significant differences are observed by nickel K-edge X-ray absorption spectroscopy between the plant and bacterial enzymes. The Ni X-ray absorption edge spectra display an 8332-eV 1s --> 3d peak intensity similar to that observed for five-coordinate Ni(II) compounds1 for both native and 2-ME-bound derivatives. Curve-fitting of Ni EXAFS data indicates that the average Ni(II) coordination environment in native urease can be described as Ni(imidazole)x(N,O)5-x, with x = 2 or 3. Addition of 2-ME results in replacement of one of the non-imidazole (N,O) ligands with (S,Cl) (most likely the thiolate sulfur of 2-ME) and results in the appearance of a new peak in the Fourier transforms that can only be fit with a Ni...Ni scattering component at a Ni-Ni distance of approximately 3.26 angstrom. A structure for this 2-ME-bound dinuclear site is proposed to contain the two Ni(II) ions bridged by the thiolate sulfur of 2-ME.