The reaction of nitrocefin with metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was studied using rapid-scan and stopped-flow ultraviolet-visible (UV-vis) studies in an effort to discern the kinetic mechanism used by L1 to hydrolyze penicillins and cephalosporins. Rapid-scan and stopped-flow UV-vis studies of nitrocefin hydrolysis by L1 identified three species: (1) the substrate (nitrocefin) displayed an absorbance peak at 390 nm (epsilon = 11 500 M(-1) cm(-1)) that decreased during the reaction with a rate constant of 170 +/- 30 s(-1); (2) the product (hydrolyzed nitrocefin) displayed an absorbance peak at 485 nm (epsilon = 17 420 M(-1) cm(-1)) that increased during the reaction with rate constant of 40 +/- 1 s(-1); and (3) an intermediate displayed an absorbance peak at 665 nm (epsilon = 32 000 M(-1) cm(-1)) that increased initially with a rate constant of 190 +/- 3 s(-1) and then decreased with a rate constant of 38 +/- 2 s(-1). Single-turnover experiments demonstrated that there were no pre-steady-state bursts in the reaction of L1 with nitrocefin; moreover, the progress curves could be fit to a kinetic mechanism that includes the formation of a transient intermediate by using KINSIM and the rate constants given above. Progress curves from experiments conducted at different reaction conditions or with a different substrate could also be fit to the proposed kinetic mechanism. The evidence for the presence of an intermediate along with kinetic simulations supports a hydrolytic mechanism for L1 that involves an intermediate whose breakdown is rate-determining.