This study was performed to test the hypothesis that the amount of nitric oxide (NO) in exhaled air (VNO) is the net result of both NO formation in the conducting airway and its clearance by diffusion in the alveoli. It's so difficult to collect the gas in the alveoli in volunteers that we made the following consideration from the profile of CO2 fraction (FECO2): the late fraction of exhaled air coming mainly from the alveoli while the early fraction representing mixed gas from both the conducting airway and/or the transition zone, and the alveoli. We compared the FECO2, NO concentration, and VNO in the early and late fractions of exhaled air after subjects inspiring either NO-free gas or NO-containing gas (510 ppb) using a single-breath technique (n = 5). After inspiring both the NO-free and NO-containing gases, NO appeared in a significantly lower concentration and amount in the late fraction of exhaled air than those in the early fraction. If NO was not cleared by diffusion in the alveoli or the transition zone, exhaled NO in the late fraction should not differ significantly from that in the early fraction. The results suggest that: 1) NO is constantly formed in the conducting airway and/or the transition zone, and is acquired by both inhaled and exhaled air; and 2) NO is cleared by diffusion in the alveoli.