The hydrolysis of cephalosporins containing good leaving groups at the 3''-position [those used in this study were the chromogenic cephalosporin PADAC [pyridine-2-azo-4''-(N'',N''-dimethylaniline) substituted on cephalosporin], cephaloridine and cephalothin], catalyzed by the Staphylococcus aureus PC1 .beta.-lactamase, proceeds in 2 spectrophotometrically observable phases. The first involves formation of an acyl-enzyme intermediate while the second involves partitioning of this intermediate between 2 pathways. One path yields the normal cephalosporoate from which the 3''-leaving group is spontaneously eliminated in solution to give the 3-methylenedihydrothiazine 2, while the second involves initial elimination of the 3'' substituent, thus yielding a second acyl-enzyme intermediate, which than hydrolyzes to give the same final product as from the first pathway. The second acyl-enzyme is relatively inert to hydrolysis (t1/2 [half-time] .simeq. 10 min at 20.degree. C), and its formation thus leads to transient inhibition of the enzyme. The partition ratio between hydrolysis and elimination at the enzyme active site could be determined either spectrophotometrically from burst experiments or from measurements of residual .beta.-lactamase activity as a function of cephalosporin concentration. This ratio varied with the leaving group ability of the 3'' substituent (acetoxy > N,N-dimethylaniline-4-azo-2''-pyridinium > pyridinium) in the anticipated fashion. The inert acyl-enzyme intermediate was isolated by exclusion chromatography and shown to contain the cephem nucleus, but not the 3'' substituent, covalently bound to the enzyme. As would be expected, PADAC, cephaloridine and cephalothin yielded the same insert intermediate. Cephalosporins with poor or no 3''-leaving groups, e.g., dansylcephalothin and desacetoxycephalothin, neither displayed the branched pathway nor yielded the long-lived acyl-enzyme.