Temocillin, a 6-methoxy derivative of ticarcillin stable against hydrolysis by most β-lactamases including extended-spectrum β-lactamases (ESBLs) and AmpC-type β-lactamases, is a potential alternative to carbapenems for infections caused by Enterobacteriaceae producing these enzymes. Carbapenem-resistant KPC-producing Klebsiella pneumoniae and Escherichia coli have emerged, with limited treatment options including colistin (nephrotoxicity) and tigecycline (poor urinary concentration), and resistance to these agents is emerging. The present study evaluated the in vitro activity of temocillin against 33 KPC-producing clinical isolates (30 K. pneumoniae, 3 E. coli, confirmed by ertapenem resistance, modified Hodge test, and KPC PCR). MICs were determined by agar dilution. For K. pneumoniae, MICs ranged 16-64 μg/ml (MIC50 and MIC90 32 μg/ml); E. coli clinical isolates had MICs 8-16 μg/ml, and an E. coli isogenic clone producing KPC-3 had an MIC of 8 μg/ml. A mild inoculum effect (twofold MIC difference) was seen for K. pneumoniae at 10⁶ CFU, and mutation frequencies were low. Given BSAC breakpoints (systemic ≤8 μg/ml, urinary ≤32 μg/ml) and temocillin's pharmacokinetics (peak serum ~160 μg/ml, urinary ~500 μg/ml after 500 mg), temocillin is a potential alternative for mild to moderate urinary tract infections caused by KPC-producing Enterobacteriaceae.