The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.