Twenty-five years after its introduction, ceftazidime remains the most active cephalosporin against Pseudomonas aeruginosa. Nevertheless, resistance arises by upregulation of AmpC β-lactamase, by efflux or, less often, via acquisition of additional β-lactamases. Mutational resistance is especially prevalent among cystic fibrosis (CF) isolates. We examined the activity of a novel oxyimino-aminothiazolyl cephalosporin, CXA-101 (FR264205), against P. aeruginosa strains with defined resistance mechanisms as well as against multiresistant clinical CF isolates of P. aeruginosa and Burkholderia cepacia. Minimum inhibitory concentrations (MICs) of CXA-101 were determined by the Clinical and Laboratory Standards Institute agar dilution method and were 0.25–0.5 mg/L for ‘typical’ P. aeruginosa strains without acquired resistance, compared with 1–2 mg/L for ceftazidime. MICs of CXA-101 were 0.5–2 mg/L and 4 mg/L, respectively, for isolates with upregulated efflux or total AmpC derepression, compared with 2–16 mg/L and 32–128 mg/L for ceftazidime. Full activity was retained against OprD mutants resistant to imipenem. Substantive resistance (MICs ≥ 32 mg/L) arose for transconjugants with PER, VEB and OXA extended-spectrum β-lactamases and for metallo-β-lactamase producers, with reduced susceptibility (MIC = 8 mg/L) for transconjugants with OXA-2, OXA-3 and NPS-1 enzymes. MICs of CXA-101 were 2- to 16-fold below those of ceftazidime for multiresistant P. aeruginosa from CF patients, but ranged up to >128 mg/L; values for B. cepacia from CF resembled those for ceftazidime.