Techniques for collecting exhaled nitric oxide (ENO) recommend the use of antibacterial filters of 0.3 µm. The aim of the present study was to compare the measurements of ENO obtained with two different filtering devices. Air samples from 17 asthmatic and 17 non-asthmatic subjects were collected by a recommended off-line technique using two different mouthpieces: 1) the Sievers disposable tool (A) under a breathing pressure of 18 cmH2O, and 2) a mouthpiece containing a HEPA filter (B) under a breathing pressure of 12 cmH2O. The nitric oxide samples were collected into an impermeable reservoir bag. Values for ENO were compared using two-way repeated measures ANOVA followed by the Tukey test. Agreement was assessed by Bland-Altman analysis. ENO values obtained with mouthpieces A and B were comparable for asthmatic (mean ± SEM, 42.9 ± 6.9 vs 43.3 ± 6.6 ppb) and non-asthmatic (13.3 ± 1.3 vs 13.7 ± 1.1 ppb) subjects. There was a significant difference in ENO between asthmatics and non-asthmatics using either mouthpiece A (P<0.001) or B (P<0.001). There was a positive correlation between mouthpiece A and mouthpiece B for both groups. The Bland-Altman limits of agreement were considered to be acceptable. Mouthpiece B was less expensive than A, and these data show that it can be used without compromising the result. Our data confirm reports of higher ENO values in the presence of airway inflammation.