The pH at the site of infection is one of a number of factors that may significantly influence the in vivo activity of an antibiotic prescribed for treatment of infection and it may be of particular importance in the treatment of cystic fibrosis (CF) pulmonary infection, as acidification of the airways in CF patients has been reported. As Pseudomonas aeruginosa is the most frequent causative pathogen of CF pulmonary infection, this study determines the effect that growth at a reduced pH, as may be experienced by P. aeruginosa during infection of the CF lung, has on the susceptibility of clinical P. aeruginosa isolates, grown planktonically and as biofilms, to tobramycin and ceftazidime. Time-kill assays revealed a clear loss of tobramycin bactericidal activity when the isolates were grown under acidic conditions. MIC and MBC determinations also showed decreased tobramycin activity under acidic conditions, but this effect was not observed for all isolates tested. In contrast, growth of the isolates at a reduced pH had no adverse effect on the bacteriostatic and bactericidal activity of ceftazidime. When the isolates were grown as biofilms, the pH at which the biofilms were formed did not affect the bactericidal activity of either tobramycin or ceftazidime, with neither antibiotic capable of eradicating biofilms formed by the isolates at each pH. This was in spite of the fact that the concentrations of both antibiotics used were much higher than the concentrations required to kill the isolates growing planktonically. These results show that growth in an acidic environment may reduce the susceptibility of clinical P. aeruginosa isolates to tobramycin.