HMRZ-86 was designed as a new chromogenic cephalosporin to detect extended-spectrum beta-lactamases (ESBLs) and similar evolved beta-lactamases, such as metallo-beta-lactamases, derepressed AmpC, and extended oxacillinase. We report here our investigation of the kinetic parameters of several types of beta-lactamases to show the enzymatic characteristics of HMRZ-86. The Michaelis constant (Km values of HMRZ-86 for ESBLs were twice to three and half times as high as those of nitrocefin, and the maximum velocity (Vmax) was one-fifth that of nitrocefin. The Km and Vmax of HMRZ-86 for AmpC were both smaller than those of nitrocefin. The kinetic parameters of HMRZ-86 for metallo beta-lactamase (MBL) were very variable, depending on the type of buffer solution used and the concentration of zinc ions. For MBL, the Km values of HMRZ-86 were higher than those of nitrocefin, but the Vmax values were almost the same as those of nitrocefin. Although the chemical structure of HMRZ-86 is similar to that of nitrocefin, we think the enzymatic reactivities of the two entities for beta-lactamases are very different.