Acyl phosph(on)ates are shown to inhibit serine beta-lactamases and provide a new source of relatively stable complexes. Thus, benzoyl phenyl phosphate, benzoyl phenylphosphonate, and dibenzoyl phosphate react with the class C beta-lactamase of Enterobacter cloacae P99 at micromolar concentrations to form an acyl enzyme of half-life about 40 s. The phosphonate reacts further more slowly to produce a much more inert complex. Dibenzoyl phosphate reacts with the class A TEM beta-lactamase to form an acyl enzyme of half-life about 8 s and, more slowly, reaching completion after an average of about 80 turnovers, a more inert complex, of half-life about 2 h. The acyl phosphonates thus represent a new starting point for the design of beta-lactamase inhibitors and perhaps of antibacterial agents.