The ring opening of 2-azetidinone via a neutral H2O-assisted aminolysis process is studied using different quantum chemical methods (MP2/6-31G**, B3LYP/6-31G**, and G2(MP2,SVP) levels of theory) as a further step to the theoretical investigation of the aminolysis reaction of beta-lactam antibiotics (Diaz, N.; Suarez, D.; Sordo, T. L. Chem Eur. J. 1999, 5, 1045-1054; Diaz, N.; Suarez, D.; Sordo, T. L. J. Org. Chem.. 1999, 64, 9144-9152). The calculated pathways are analogous to those previously described for the amine-assisted aminolysis reaction, a syn stepwise mechanism being the most favored one both in the gas phase and in solution with an energy barrier of around 39.7 kcal/mol (Delta G(solution)). For this process the strong catalytic action of one water molecule amounts to 23.0 (G2(MP2,SVP) electronic energy), 12.6 (Delta G(gas-phase)), and 9.1 kcal/mol (Delta G(solution)). The water-assisted reaction between methylamine and the 3 alpha-carboxypenam anion was also studied at the B3LYP/6-31+G* level of theory to model the specific role of water in the aminolysis of bicyclic beta-lactam antibiotics. For this system the most favorable attack of CH3NH2 to the carbonylic group in a low polar media can occur through simultaneous H-transfer to the carboxylate group assisted by the ancillary water molecule with a Delta G(gas phase) barrier of 31.0 kcal/mol with respect to separate reactants. A concerted route becomes the most favored mechanism for the aminolysis of penicillins assisted by an ancillary water molecule in aqueous solution. The structure of the concerted transition stare is in agreement with the experimentally reported Bronsted beta-values close to unity. Interestingly, according to the experimental structure of the Lys(199) active center of Human Serum Albumin, the mechanism for the aminblysis reaction of beta-lactams in a protein environment may be similar to the stepwise route found in the gas phase for 3 alpha-carboxypenam.