Colonization of the airways by filamentous fungi can occur in asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. A recent study found IgE sensitization to Aspergillus fumigatus to be associated with reduced lung function. Significantly higher rates of A. fumigatus were detected in sputum from asthmatics sensitized to this fungus compared to non-sensitized asthmatics. The rate of positive cultures was far higher than equivalent historical samples analysed by the local clinical laboratory following protocols recommended by the UK Health Protection Agency (HPA). This study compares the HPA procedure with our sputum processing method, whereby sputum plugs are separated from saliva and aliquots of approximately 150 mg are inoculated directly onto potato dextrose agar. A total of 55 sputum samples from 41 patients with COPD were analyzed, comparing fungal recovery of five dilutions of sputa on two media. Isolation of A. fumigatus in culture was significantly higher using the research approach compared to the HPA standard method for mycological investigations (P < 0.001). There was also a significant difference in the recovery rate of A. fumigatus (P < 0.05) between media. This highlights the need for a standardized approach to fungal detection which is more sensitive than the method recommended by the HPA.