At the Danish CF Center patients with chronic Pseudomonas aeruginosa lung infection were treated 3-4 times a year (from 1976) with a 2-week intravenous antipseudomonal course which included preferentially an aminoglycoside and a beta-lactam antibiotic. We investigated the development of antibiotic resistance in P. aeruginosa strains isolated from Danish CF patients over a period of 18 years by testing the in vitro efficacy of carbenicillin, piperacillin, ceftazidime, tobramycin and ciprofloxacin against P. aeruginosa strains collected in 1973 (51 strains), 1980 (80 strains), 1985 (58 strains), and 1991 (100 strains). All the strains were screened and assayed semiquantitatively for beta-lactamase activity by use of nitrocefin. We found a significant (p < 0.005) increase in the MIC values of the P. aeruginosa strains against piperacillin and ceftazidime. However, no significant correlation was found between the MIC and the number of antipseudomonal courses of antibiotics. The proportion of resistant in vivo selected P. aeruginosa strains, presumed to be stably derepressed producers of chromosomal beta-lactamase, also increased significantly during the period studied. Our results confirm that the beta-lactamase production is an important mechanism of antibiotic resistance in P. aeruginosa.