Many markers of airway inflammation and oxidative stress can be measured non-invasively in exhaled breath condensate (EBC). However, no attempt has been made to directly detect free radicals using electron paramagnetic resonance (EPR) spectroscopy. Condensate was collected in 14 children with cystic fibrosis (CF) and seven healthy subjects. Free radicals were trapped by 5,5-dimethyl-1-pyrroline-N-oxide. EPR spectra were recorded using a Bruker EMX spectrometer. Secondly, to study the source of oxygen centered radical formation, catalase or hydrogen peroxide was added to the condensate. Radicals were detected in 18 out of 21 condensate samples. Analysis of spectra indicated that both oxygen and carbon centered radicals were trapped. Within-subject reproducibility was good in all but one subject. Quantitatively, there was a trend towards higher maximal peak heights of both oxygen and carbon centered radicals in the children with CF. Catalase completely suppressed the signals in condensate. Addition of hydrogen peroxide resulted in increased radical signal intensity. Detection of free radicals in EBC of children with CF and healthy subjects is feasible using EPR spectroscopy.