TEM beta-lactamase variants with the amino acid substitutions R164S, E104K, G238S, and E240K (ABL numbering) display increased activity toward extended-spectrum cephalosporins. The T265M substitution is frequently found to be associated with the above substitutions in extended-spectrum beta-lactamases. However, the residue is located away from the active site in the three-dimensional structure and has been assumed to have no effect on catalysis. To examine the effect of the substitution on the structure and function of TEM beta-lactamase we constructed the following mutants: G238S, T265M, T265M:G238S, and T265M:G238S:E240K. Each enzyme was purified to homogeneity and the kinetic parameters kcat, Km and kcat/Km were determined for cefotaxime, ceftazidime, cephaloridine, and ampicillin. The results indicate that the T265M mutation has little effect on hydrolysis. In addition, we used immunoblotting to show that the substitution has little or no effect on the in vivo steady-state levels of beta-lactamase.