The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more pronounced in the R244C mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes.