Measurement of exhaled nitric oxide (NO) may allow noninvasive assessment of inflammatory disease in the lung. We determined immediate and day-to-day reproducibility of single-breath NO measurements at different points on the exhaled test, and whether levels recorded reflect levels of NO in the lower airways. Using a rapid chemiluminescence analyser, 55 healthy control subjects performed three sequential tests on each of two days. NO levels were compared at the level corresponding with: 1) the time the mouth pressure fell below 4 cmH2O (MP); 2) the plateau of end-exhaled CO2 (CO2); and 3) the NO plateau (NOp). NO levels were measured directly from the lower airways of 15 lung transplant recipients and compared with NO levels from a single-breath test performed in the same cohort. For measurements performed at MP, CO2 and NOp, the mean +/- SD differences between the two closest levels performed on the same day were 0.11+/-0.18, 0.095+/-0.16 and 0.094+/-0.13 parts per billion (ppb), respectively, and between days were 0.18+/-0.76, 0.19+/-0.78 and 0.17+/-0.8 ppb, respectively. End-expiratory levels recorded at the mouth from a single-breath test and in the lower airways were highly correlated (mouth versus trachea r2=0.95, p<0.0001, mouth versus bronchus r2=0.92, p<0.0001). Single-breath exhaled nitric oxide levels are a simple, reproducible and valid measure of nitric oxide production from the lower respiratory tract.