Nasal nitric oxide measurement may be a surrogate marker of upper airway inflammation. There is, however, no standardized measurement technique; and this led us to examine measurement techniques for acceptability and reproducibility. In five subjects we examined the flow dependence of nasal NO. In 13 healthy volunteers, nasal NO was measured on-line by five methods: 1) Tidal nasal and oral breathing: NO sampling during exclusive nasal followed by exclusive oral tidal breathing; 2) Fixed flow exhalation: NO sampling during exclusive nasal followed by exclusive oral exhalation at 100 mL/second from total lung capacity; 3) Nasal-oral aspiration: air aspirated from the mouth via both nares at 100 mL/second with glottis closure; 4) Aspiration from one nares: air aspirated from one nares at 3.3 mL/second using nitric oxide analyzer sample line with velum closure; 5) Nasal Insufflation: NO sampled at one nares as air insufflated into the other nares at a flow of 100 mL/second with velum closure. Acceptability of all methods was assessed by subjects and technicians. Nasal NO concentration showed a significant inverse correlation with transnasal flow rate. All methods showed excellent reproducibility as assessed by the intraclass correlation coefficient except tidal breathing, which showed highly variable breath-to-breath NO levels, although mean breath values were reproducible. Mean nasal NO concentrations with methods 1, 2, 3, 4, and 5 were 32.1, 50.2, 62.8, 1381, and 60.0 ppb, respectively. Velum closure was not always achieved in methods 4 and 5, whereas methods 1 and 2 required separate nasal and oral procedures. Method 5 had reduced acceptability. NO concentrations were similar with methods that used the same airflow (2, 3, and 5). Nasal NO can be sampled in different ways with excellent reproducibility. In view of the flow dependence of nasal NO, it is vital to use a constant flow rate, and lower airway NO contribution must be excluded or subtracted. The fixed flow exhalation appears to be the preferred method as it is highly reproducible and acceptable.