Different penicillin-binding proteins PBPs are affected in cefotaxime-resistant laboratory mutants compared to piperacillin-resistant mutants. PBP2x acts as the primary PBP target in cefotaxime-resistant mutants, whereas PBP2b is the primary target in piperacillin-resistant mutants. Depending on the mutations in PBP2x, it functions as a resistance determinant for cefotaxime only, or for penicillins as well. Mutations in PBP2x of laboratory mutants are found exclusively in the penicillin-binding domain that contains three homology boxes common to all penicillin-interacting enzymes. Most mutations relevant for resistance occur close to the SXN or the KT/SG box, or at the C-terminal end of the penicillin-binding domain, similar to mutations described in PBP2b of laboratory mutants. Amino acid alterations occur at similar sites also in PBP2x of beta-lactam-resistant clinical isolates and most of these proteins also contain changes in the SXXK box with the active site serine, suggesting that these alterations may be critical for resistance development in clinical isolates.