A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (class A and C) beta-lactamases. Variations of the leaving group in a series of methyl phosphonates showed that leaving groups better than the previously employed p-nitrophenoxide could give more effective inhibitors. Inclusion of a negative charge in the leaving group did not, per se, lead to better inhibitors. Aryl phosphonates appeared more effective than those with electronically comparable but smaller leaving groups. The combination of a good leaving group, 2,4-dinitrophenoxide, with an amido side-chain, phenylmethylsulfonamido--the latter rather than phenylacetamido in order to increase the stability of the compound with respect to intramolecular nucleophilic catalysis of hydrolysis by the amide group--did not yield overall a better inhibitor than previously employed p-nitrophenyl phosphonates. These results give the first indication of specific interactions between a beta-lactamase and the leaving group of a phosphonate inhibitor. Only one enantiomer of a chiral thiophosphonate, presumably the Rp isomer, was an effective inhibitor. Addition of either a D- or a L-methyl group to the methylene group of a p-nitrophenyl amidomethylphosphonate did not enhance the inhibitory ability of the phosphonate. Class A beta-lactamases remain refractory to phosphonates.