Five methods were tested by RAST for allergen extraction from seven basidiospore species Armillaria tabescens, Chlorophyllum molybdites, Coprinus quadrifidus, Pleurotus ostreatus, Calvatia cyathiformis, Pisolithus tinctorius, and Scleroderma sp. With each basidiospore type, extracted allergen activity varied according to the method employed. In general, defatting of spores with ethyl ether, followed by homogenization in 0.125 m NH4HCO3 buffer, resulted in greatest allergen yield. In order to compare extracts from different isolates, batches of Pleurotus ostreatus spores obtained from different locations and over different time periods were analysed. Spores harvested from the same basidiomycete species in different areas varied in allergen (RAST) and protein (HPLC profile) content. The spores obtained from the same location over a 1–2 year period did not differ significantly. These results indicate that basidiospores can be stored for several years and that spore extracts from different locations can vary. These studies will help in the future to provide better characterized extracts for clinical studies.