The emergence of beta-lactam-resistant strains of Pseudomonas aeruginosa in a cystic fibrosis patient treated with high-dose tobramycin and piperacillin was studied. Two serotypes, M and K, were present before treatment and persisted, with changes in their beta-lactam resistance spectra, during treatment. The resistance was correlated with changes in the penicillin-binding proteins (PBPs) in both serotypes. In the low-level-resistant serotype K organism, PBP-3 either was absent or had lost the ability to bind [14C]penicillin G. Tow serotypes M strains, one with low- and one with high-level resistance to several antipseudomonal beta-lactam antibiotics, were isolated at progressively later stages of therapy. Several differences were noted between the PBP patterns of the resistant M and the susceptible M strains. The affinity for [14C]penicillin G was reduced in both resistant strains. PBP bands, with the exception of PBP-6 in the most resistant M type, were barely or not detectable at a [14C]penicillin G concentration of 39 microgram/ml. The graduated decrease in affinity for [14c]penicillin G was correlated with increasing beta-lactam resistance and with an increase in the quantity of the protein corresponding to PBP-6. The emergence of the low-level-resistant strains midway through, and of the highly resistant strain in the final stages of, the reported treatment strongly suggested that the resistance resulted from mutation in those strains present before treatment selected for by the high-dose piperacillin treatment.