CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. We evaluated the activity of CXA-101 against a highly challenging collection of beta-lactam-resistant Pseudomonas aeruginosa mutants selected in vitro and after antipseudomonal treatment of intensive care unit (ICU) patients. The in vitro mutants investigated included strains with multiple combinations of mutations leading to several degrees of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). CXA-101 remained active against even the AmpD-PBP4 double mutant (MIC = 2 microg/ml), which shows extremely high levels of AmpC expression. Indeed, this mutant showed high-level resistance to all tested beta-lactams, except carbapenems, including piperacillin-tazobactam (PTZ), aztreonam (ATM), ceftazidime (CAZ), and cefepime (FEP), a cephalosporin considered to be relatively stable against hydrolysis by AmpC. Moreover, CXA-101 was the only beta-lactam tested (including the carbapenems imipenem [IMP] and meropenem [MER]) that remained fully active against the OprD-AmpD and OprD-PBP4 double mutants (MIC = 0.5 microg/ml). Additionally, we tested a collection of 50 sequential isolates that were susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones that emerged during treatment of ICU patients. All of the mutants resistant to CAZ, FEP, PTZ, IMP, MER, or ciprofloxacin showed relatively low CXA-101 MICs (range, 0.12 to 4 microg/ml; mean, 1 to 2 microg/ml). CXA-101 MICs of pan-beta-lactam-resistant strains ranged from 1 to 4 microg/ml (mean, 2.5 microg/ml). As described for the in vitro mutants, CXA-101 retained activity against the natural AmpD-PBP4 double mutants, even when these exhibited additional overexpression of the MexAB-OprM efflux pump. Therefore, clinical trials are needed to evaluate the usefulness of CXA-101 for the treatment of P. aeruginosa nosocomial infections, particularly those caused by multidrug-resistant isolates that emerge during antipseudomonal treatments.