The mechanistic pathway followed by the E. coli R TEM ß-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the ß-lactamase-catalysed hydrolysis of the 7-a-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known ß-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a ß-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N -acyl derivatives of 6-ß-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the ß-lactamase.