Exhaled nitric oxide (ENO) is a noninvasive marker of airway inflammation. The purpose of this study was to compare a standardized offline ENO measurement apparatus with a validated on-line method. Asthmatic volunteers (n = 21) had ENO measured by the two following methods: (1) inhalation to total lung capacity (TLC) followed by exhalation at a constant flow (45 mL/s) against a high resistance, while monitoring nitric oxide (NO) and pressure on-line; and (2) inhalation to TLC and exhalation into mylar balloons via an apparatus that included the same resistance and flow rate as used in the on-line method. We also examined NO stability in mylar balloons over 48 h. ENO values (given as geometric mean in parts per billion [ppb]; 95% confidence intervals) differed between the on-line method (69.6; 42.6 to 113.8) and the offline method (49.5; 30.9 to 79.3), indicating that the offline method gave lower ENO measures than the on-line method (p < 0.001). Furthermore, this difference between measures increased with increasing mean values. The intraclass correlation coefficient (0.931), however, showed excellent correlation between the on-line and offline methods. Within-subject repeatability, as assessed by the coefficient of repeatability (CR), was good for both the on-line and offline methods (CR, 1.09 and 1.17, respectively). Geometric mean NO concentrations (95% confidence limits) in mylar balloons containing exhalate increased from a baseline of 55.8 ppb (36.9, 84.4) to 64.5 ppb (45.6, 91.1) and 69.5 ppb (51.4, 94.0) at 24 h and 48 h, respectively. The offline method gave reproducible ENO values that were consistently smaller than, but showed good correlation with, values obtained with on-line ENO collection. This method is suitable for offline collection, but the measured values are not interchangeable with those obtained by on-line measurement.