The bacterial pathogen Pseudomonas aeruginosainfects the lungs of patients with cystic fibrosis, causing a decline in lung function and a reduction in life expectancy. The physiology of P. aeruginosahas been well studied in laboratory culture but not during infection in patients, where it may exist under anoxic conditions. Gene expression in bacteria is responsive to environmental conditions. We have taken advantage of regulated changes to gene expression to investigate the conditions experienced by the bacteria during infection. We isolatedP. aeruginosafrom sputum from the lungs of 20 patients with cystic fibrosis and used RT-qPCR to measure expression of three genes narI, nirSand ccoNthat encode proteins required for growth of P. aeruginosawhen oxygen tension is low. Expression of all three genes was significantly increased (median increases of 6-fold, 14-fold and 200-fold for ccoN, narI and nirS) when the bacteria were grown in the absence of oxygen. In parallel RNA was extracted directly from sputum from the same patients, with no bacterial subculture, and used RT-qPCR to obtain a snapshot of bacterial gene during infection. Expression of the target genes was at levels similar to those of bacteria grown in the absence of oxygen in laboratory culture. Our findings show that during chronic lung infection P. aeruginosaexists under conditions of oxygen deprivation. This finding has implications for the effectiveness of antibiotics, which we have shown have altered activity against bacteria grown in the absence of oxygen.