Inactivation of beta-lactam antibiotics by metallo-beta-lactamase enzymes is a well-recognized pathway of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a beta-lactam substrate nitrocefin (1) catalyzed by dinuclear zinc(II) model complexes was investigated in nonaqueous solutions. The initial step involves monodentate coordination of the nitrocefin carboxylate group to the dizinc center. The coordinated substrate is then attacked intramolecularly by the bridging hydroxide to give a novel intermediate (2') characterized by its prominent absorbance maximum at 640 nm, which affords a blue color. The NMR and IR spectroscopic data of 2' are consistent with it being zinc(II)-bound N-deprotonated hydrolyzed nitrocefin that forms from the tetrahedral intermediate upon C-N bond cleavage. Protonation of the leaving group is the rate-limiting step in DMSO solution and occurs after the C-N bond-breaking step. Addition of strong acids results in rapid conversion of 2' into hydrolyzed nitrocefin (3). The latter can be converted back to the blue species (2') upon addition of base. The low pK(a) value for the amino group in hydrolyzed nitrocefin is explained by its involvement in extended conjugation and by coordination to zinc(II). The blue intermediate (2') in the model system resembles well that in the enzymatic system, judging by its optical properties. The greater stability of the intermediate in the model, however, allowed its characterization by (13)C NMR and infrared, as well as electronic, spectroscopy.