One hallmark of COPD is colonization and infection of the lung. Acute exacerbations of COPD (AECOPD) are acute deteriorations of the chronic disease and are associated with a change of the pulmonary microbial balance. The collection of exhaled breath condensate (EBC) can be used to non-invasively determine markers of lung disease. The aim of the present study was to compare the results of assays based on the detection of microbial nucleic acids from EBC and from spontaneous sputum in patients with AECOPD. EBC and sputa of 29 adults with AECOPD were obtained. Isolated DNA or RNA were used as starting material for the PCR assays to detect Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, influenza viruses (AH 1, AH 3) and respiratory syncytial virus. Bacterial or viral nucleic acids were identified in 14 EBC and 21 sputa from 29 patients. Results from EBC did not correlate well with those from sputum. Viral and S. pneumoniae nucleic acids were detected only in sputum, whereas L. pneumophila DNA was only found in EBC. In three EBC and 10 sputa nucleic acids of more than one microorganism was detected. Bacterial nucleic acids can be identified in EBC of COPD patients with exacerbations. The results obtained from EBC and sputum did not correlate well. © 2011 The Authors. Respirology © 2011 Asian Pacific Society of Respirology.