Multiresistant Pseudomonas aeruginosa isolates can chronically infect patients with cystic fibrosis. Acute infective exacerbations are treated with combinations of two antipseudomonal antibiotics. Patients may respond clinically even if the bacteria are resistant, possibly due to antimicrobial synergy. The challenge for testing for synergy in vitro is that there is no standardized method, and the antibiotic susceptibility in a population of P. aeruginosa isolates in a single sputum sample can vary. We therefore compared (i) antibiotic combinations with different examples of resistant bacteria from the same sputum sample and (ii) the results of synergy testing by different methods. Antibiotic synergy was tested by using resistant P. aeruginosa isolates recovered from sputum samples taken just before the start of treatment for an acute infective exacerbation. Several examples of each morphotype of P. aeruginosa were tested by cidal checkerboard, time-kill curve, and multiple-combination bactericidal testing. The isolates were typed by pulsed-field gel electrophoresis (PFGE). The results were compared with the clinical and microbiological responses to 14 days of antibiotic treatment. Forty-four resistant isolates from nine patients were tested. Some P. aeruginosa isolates with the same morphotype and PFGE pulsotype had different results by synergy testing. There was a poor correlation between the results of the different methods of synergy testing, and no one method would have predicted the response to treatment in all patients. The in vitro effects of antibiotic combinations against different isolates from the same sputum sample can vary, and the results depend on the methodology used. The role of combination testing for the treatment of antibiotic-resistant P. aeruginosa in acute exacerbations of chronic infection in patients with cystic fibrosis needs to be reviewed.