We studied the genetic origins of piperacillin-tazobactam resistance among nosocomial Klebsiella pneumoniae strains. A total of 30 nosocomial isolates resistant to piperacillin-tazobactam were obtained from various regions of Turkey. Isoelectric focusing demonstrated at least 2 enzymes common to all strains: I at a pI of 8.0 and the other at 5.4. Piperacillin-tazobactam resistance was successfully transferred from all of the strains to Escherichia coli. Of the piperacillin-tazobactam-resistant transconjugates, 23 were also resistant to ceftazidime. However, 7 transconjugates were susceptible to ceftazidime but resistant to piperacillin-tazobactam, producing a single enzyme focusing at pI 5.4. Piperacillin resistance caused by this enzyme was reversed by clavulanate and by increased amounts of tazobactam, which indicates that this enzyme confers resistance due to its high amount. Sequence analysis revealed this enzyme to be TEM-1. This study demonstrates that transferable hyper-produced TEM-1 causes piperacillin-tazobactam resistance in Klebsiella strains in Turkish hospitals.