Penicillanic acid sulfone (1) is both a substrate and an inactivator of the RTEM beta-lactamase. About 7000 hydrolytic events occur before enzyme inactivation. The 6,6-dideuterio sulfone shows a 3-fold acceleration of both the hydrolysis reaction and the enzyme inactivation. The kinetic and spectroscopic results are nicely accommodated by a scheme in which a transiently stable intermediate is formed in an isotopically sensitive step. The deuterated material partitions less readily toward this transiently stable intermediate by virtue of a primary isotope effect, and more enzyme is then available for the hydrolysis and inactivation pathways. Use of the stereospecifically monodeuterated sulfones shows that the 6 beta hydrogen is preferentially abstracted in the formation of the transiently stable intermediate and allows a detailed picture of the interaction of the sulfone and the beta-lactamase to be drawn. The crystal structures of both the labeled and unlabeled compounds are reported.