The development of significant mechanisms of resistance to beta-lactam antibiotics in Pseudomonas aeruginosa in cystic fibrosis (CF) patients have been studied in ten CF patients during a two week course of anti-pseudomonal beta-lactam antibiotic therapy. Sputum samples were collected on days 1, 7 and 15. Entire homogenized sputum samples were examined directly for the number of bacteria resistant to different levels of antibiotics. This allowed the detection of pre-existing resistant subpopulations of bacteria as well as following the changes in beta-lactam antibiotic susceptibility during treatment. P. aeruginosa isolates were characterized by means of sero-grouping, phage- and pyocin-typing. Outer membrane proteins of paired sensitive and resistant strains were characterized. Sonicated extracts of cells were assayed for basal and induced beta-lactamase activity. Beta-lactamase activity was further characterized by isoelectric focusing and inhibition profiles. Our observations were in accordance with the hypothesis that the sensitive inducible population was overrun by the pre-existing resistant subpopulation, during treatment. The resistant in-vivo selected P. aeruginosa population exhibited stable partially derepression but the beta-lactamase inhibitor tazobactam restored beta-lactam antibiotic activity.