The specifically mutated enzyme thiol beta-lactamase has been expressed in Escherichia coli by means of the trp promoter and purified to homogeneity. The gene for this enzyme results from a single base change N410 A----T in the gene of pBR322 RTEM beta-lactamase (EC 126.96.36.199, penicillinase, penicillin amido-beta-lactamhydrolase) which alters the codon for the active site Ser 70 to that for Cys. Precursor thiol beta-lactamase is processed to give the same NH2-terminal sequence as that for wild type enzyme. In contrast to the wild type enzyme, thiol beta-lactamase contains one free titratable thiol group/molecule. Thiol beta-lactamase catalyzes the hydrolysis of beta-lactams with a substrate specificity that is distinct from that of wild type enzyme. For benzyl-penicillin and ampicillin, the Km values are similar to wild type values although the kcat values are 1-2% that of wild type enzyme. For the cephalosporin nitrocefin, the Km is greater than 10-fold that of the wild type and the kcat is at least as large as the kcat for the wild type enzyme. Thiol beta-lactamase is different from wild type beta-lactamase in that it is not competitively inhibited by boric acid although a small degree of noncompetitive inhibition does occur. Whereas the circular dichroism spectra of both enzymes are nearly identical, thiol beta-lactamase at 40 degrees C is 3-fold more resistant to trypsin than is the wild type enzyme.