A novel carbapenem-hydrolyzing β-lactamase, KPC-1, was identified in a carbapenem-resistant strain of Klebsiella pneumoniae. The enzyme, encoded on a nonconjugative 50-kb plasmid, was responsible for resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. KPC-1 showed 45% amino acid identity to Sme-1 from Serratia marcescens. It hydrolyzed carbapenems, penicillins, cephalosporins, and monobactams, with the highest affinity for meropenem. KPC-1 was inhibited by clavulanic acid and tazobactam. The strain lacked detectable levels of OmpK35 and OmpK37, which are porins involved in antibiotic resistance. The study concluded that carbapenem resistance in K. pneumoniae strain 1534 was mainly due to the production of KPC-1, a novel class A β-lactamase. The absence of OmpK35 was consistent with clinical isolates harboring class A β-lactamases. The study highlights the importance of understanding carbapenem resistance mechanisms to optimize treatment strategies.A novel carbapenem-hydrolyzing β-lactamase, KPC-1, was identified in a carbapenem-resistant strain of Klebsiella pneumoniae. The enzyme, encoded on a nonconjugative 50-kb plasmid, was responsible for resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. KPC-1 showed 45% amino acid identity to Sme-1 from Serratia marcescens. It hydrolyzed carbapenems, penicillins, cephalosporins, and monobactams, with the highest affinity for meropenem. KPC-1 was inhibited by clavulanic acid and tazobactam. The strain lacked detectable levels of OmpK35 and OmpK37, which are porins involved in antibiotic resistance. The study concluded that carbapenem resistance in K. pneumoniae strain 1534 was mainly due to the production of KPC-1, a novel class A β-lactamase. The absence of OmpK35 was consistent with clinical isolates harboring class A β-lactamases. The study highlights the importance of understanding carbapenem resistance mechanisms to optimize treatment strategies.