This report describes methods for measuring nitric oxide production by the lungs' lower alveolar airways (VNO), defined as those alveoli and bronchioles well perfused by the pulmonary circulation. Breath holding or vigorous rebreathing for 15-20 s minimizes removal of NO from the lower airways and results in a constant partial pressure of NO in the lower airways (PL). Then the amount of NO diffusing into the perfusing blood will be the pulmonary diffusing capacity for NO (DNO) multiplied by PL and by mass balance equals VNO, or VNO = DNO(PL). To measure PL, 10 normal subjects breath held for 20 s followed by exhalation at a constant flow rate of 0.83 +/- 0.14 (SD) l/s or rebreathed at 59 +/- 15 l/min for 20 s while NO was continuously measured at the mouth. DNO was estimated to equal five times the single-breath carbon monoxide diffusing capacity. By using breath holding, PL equaled 2.9 +/- 0.8 mmHg x 10(-6) and VNO equaled 0.39 +/- 0.12 microl/min. During rebreathing PL equaled 2.3 +/- 0.6 mmHg x 10(-6) and VNO equaled 0.29 +/- 0.11 microl/min. Measurements of NO at the mouth during rapid, constant exhalation after breath holding for 20 s or during rebreathing provide reproducible methods for measuring VNO in humans.