Background: Exhaled breath condensate (EBC) analysis is a promising new method to monitor airway inflammation, however there are still multiple open methodological questions. The purpose of this study was therefore to investigate methodological influences on nitrite in EBC (surface contamination, flow dependency, storage time and inter-and within day variability). Material/Methods: EBC samples of 10 healthy children, 10 healtly adults and 71 asthmatic children were collected and nitrite was analysed using the Griess reaction. Collector devices where either air-dried after disinfection or stored in distilled water till usage. Repeatability measurements were performed at three different times during one day and on five consecutive days. Flow dependency was analysed at 200 and 400 ml/s expiratory flow and storage stability was investigated at several time points within the first three hours after collection of the sample. In a preliminary study EBC nitrite was analysed in asthmatic children of different severity. Results: Surface contamination appears to have a profound influence on nitrite levels and both within-day and inter-day variability is high. We found no flow dependency, and storage stability was satisfying although with considerable variety in several samples. There were no significant differences between the nitrite levels of the different asthmatic subgroups nor between the asthmatic children and the control. Conclusions: These data indicate that EBC nitrite is a substance with a wide variety of influencing factors and different sources of origin. This has to be kept in mind when using exhaled nitrite as a biomarker for airway inflammation.