Armillaria ostoyae is a phytopathogen infecting coniferous trees. Fruiting bodies of this basidiomycete contain high phospholipase A(1) (PLA(1)) activity. In this paper, the role of phospholipid-deacylating activity, which was also detected in fruiting bodies of other basidiomycetes, in the fungal lipid metabolism is elucidated. For A. ostoyae the occurrence of PLA(1) activity is shown to be restricted to the late reproductive phase, correlating with the release of mature spores. Specific expression in the spore-producing tissue provides evidence for the involvement of PLA(1) in spore formation. Based on lipid analysis, the degradation of membrane phospholipids in this tissue can be ascribed mainly to PLA(1) activity because other enzymes such as phospholipases C and D, triglyceride lipase and phosphatidic acid phosphatase had only low activities. A concomitant increase in the concentration of fatty acids and their anabolites (di- and triglycerides), which are used as storage lipids in the developing fungal spore cells, was observed. Therefore, PLA(1) contributes to the formation of spores by providing membrane constituents as a source of fatty acids.