Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 μM) of nitrate (NO−3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO−3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 μM NO−3 and 100 μM NO−3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at