The concentration of exhaled nitric oxide (eNO) is a useful marker of asthmatic bronchial inflammation. eNO can now be measured away from the laboratory (off-line), even in children. Short exhalation maneuvers (8 sec) and small samples (1 L) of exhaled gas are probably sufficient in children, but more information is needed about the effect of different measurement conditions. As a preliminary step before conducting epidemiological studies in schoolchildren, we investigated the effects of expiratory flow, dead space, and expiratory time on eNO concentrations collected in 1-L mylar collection bags. We studied 101 cooperative subjects (62 males) aged 5–18 years (30 healthy volunteers, 51 asthmatics, and 20 children with various other respiratory diseases) in our pulmonary function laboratory. On-line and off-line eNO were compared in a single session, and analyzed with a Sievers NOA™ 280 nitric oxide analyzer. For both methods of collecting expired gas, subjects did a single exhalation without breath-holding against an expiratory pressure 10 cm H2O. We investigated the effects of expiratory flow, dead space, and exhalation time on eNO; we also compared on-line and off-line eNO measurements, and the repeatability of both techniques at a given flow rate. Expiratory flows of 58 mL/sec provided more reproducible data than lower flows (coefficient of repeatability 1.1 ppb for 58 mL/sec vs. 2.8 for 27 mL/sec vs. 5.7 for 18 mL/sec). eNO concentrations were about 25% higher in off-line than in on-line recordings if the initial 250 mL of exhaled gas were not eliminated, and 37% higher if exhalation lasted longer (16 sec vs. 8 sec). Eliminating 250 mL of dead space and shortening the filling time to 8 sec yielded off-line eNO values close to those on-line (geometric mean off-line eNO 14.4 ppb, 95% confidence interval: 12.2–17.0) vs. on-line eNO 13.8 ppb (95% confidence interval: 11.6–16.5). On-line and off-line results were highly correlated (r = 0.996, P = 0.000) and had similar coefficients of variation (on-line eNO 2.6%, off-line 2.8%). Neither agreement nor repeatability of eNO measurements were affected by disease status or baseline FEV1 (% predicted values). Once standardized, the off-line eNO technique using 1-L gas collection bags will provide results similar to those recorded on-line. Pediatr Pulmonol. 2001; 32:159–167. © 2001 Wiley-Liss, Inc.