The maneuver of breath-holding had been reported to increase the levels of eNO.4–5,9–10,15 In a study insufflating an indicator gas to the nose,17 however, breath-holding itself did not cause any gas leak from the nose to the airway. Silkoff et al16 also confirmed that breath-holding does not allow nasal contamination. In the present study, we confirmed that during breath-holding for 10 s, no gas leak from the nose to the airway occurred. Persson et al15 reported that breath-holding increases the peak eNO in a duration-dependent manner. Massaro et al9 found a significant increase in eNO after breath-holding in patients with endotracheal intubation. Theoretically, a diffusion of NO occurs both during breathing and breath-holding in the airway.7,22–23,25 We studied the effects of breath-holding on eNO levels for 10 s alone. For a more quantitative study, we should have examined the breath-holding for longer periods. In the present study, breath-holding increases the peak eNO similarly in normal subjects and asthmatic patients, but does not change the plateau eNO. When the expiratory flow is fast, the plateau eNO level is determined primarily by NO levels of the alveolar gas (phase 3), irrespective of breath-holding time.7 When the expiratory flow rate is slow, phase 3 gas passes through the airway for a longer time, resulting in a substantial amount of airway NO possibly diffusing into the exhaling air, thus increasing eNO levels. In our normal subjects, the peak eNO level after breath-holding was 54 ppb, which is very close to the equilibrium concentration (56 ppb) in trachea during breath-holding reported by DuBois et al.7 The magnitude of the increase in peak eNO in response to breath-holding in our asthmatic patients was comparable to that of normal subjects, although no change in the plateau eNO was observed. During breath-holding, NO in the airway wall may diffuse into the exhaling gas, but after breath-holding, alveolar gas (phase 3) may pass through the airway for a given time, suggesting that the amount of NO diffusing from the airway may depend on the flow rate alone.