The occurrence of a novel phospholipid, pyrophosphatidic acid, in the lipid extracts of yeasts (23 species), bacteria (E. coli), algae (chlorella), mammalia (human, rabbit, guinea pig, and mouse), insect (cockroach), fish (carp), mollusc (clam), and sperma-tophyte (spinach) was investigated. Pyrophosphatidic acid was found exclusively in the lipid extracts of several kinds of yeast species, but not in other normal living species (animals, plants, and microorganisms) so far investigated. All of the yeast species containing this lipid belong to the asporogenous yeasts (Cryptococcus neofor-mans CBS-132, Cryptococcus laurentii Z 6-5, Rhodotorula glutinis H 3-9-1, Rhodotorula rubra AY-2, Kloeckera apiculata KK-3, and Trichosporon cutaneum KC 4-3), and bal-listosporogenous yeast (Sporobolomyces salmonicolor WF 174). In contrast, no detectable amount of pyrophosphatidic acid was found in the cellular lipids of ascosporo-genous yeasts.