Conventional methods for detecting airborne fungal spores rely on either optical identification or culturing and can be time consuming or unreliable. A method for purifying DNA from conventional spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. Experiments were done using Penicillium roqueforti. As few as 10 spores could be detected in the PCR and P. roqueforti spores were detected in a background of spores of six other unrelated species. The method successfully detected P. roqueforti spores collected by rotating arm and Hirst-type spore traps in wind tunnel tests. The tests suggested that the detection limit was about 10 spores or less in the PCR. Fungal spores were also detected in air samples collected in Mexico City using fungal consensus primers, with a detection limit of about 200 spores in the PCR. The potential for using PCR-assays in conjunction with impactor samplers is discussed.