Using a random combinatorial mutagenesis of TEM beta-lactamase, directed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S. Additionally, by using cloning strategies and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E104K/G238S, which broaden the specificity of clinically isolated TEM beta-lactamase variants. The kinetic characterization of the purified double mutant E104M/G238S and its single counterparts E104M and G238S was carried out. The single mutant E104M exhibited increased kcat values against all substrates tested. Km values remained similar to the values shown by the wild-type enzyme. The mutation at E104M was responsible for the increased hydrolysis rate against cefuroxime shown by the double mutant E104/G238S. The effect of mutation G238S varied more pronouncedly, depending on the substrate. In general, a lower Km was observed, but also a decreased kcat. The double mutant E104M/G238S exhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations. We observed nearly a 1000-fold greater kcat/Km for the double mutant than for the wild type. This improvement in catalysis was the consequence of increased kcat and decreased Km values. Computed contact interactions from modeling substrate complexes show reliable results only for benzylpenicillin. The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants. Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the single and double mutants, showed that the quantitative effect of a second mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.