The ether lipids of several thermophilic archaea (archaeobacteria) were compared by negative-ion fast atom bombardment mass spectrometry. The major polar lipids in extracts ofPyrococcus furiosuswere assigned as archaeol lipids (phosphatidylglycerol diether,m/z805; phosphatidylinositol diether,m/z893; and diglycosyl diether,m/z975) and caldarchaeol lipids (diglycosyl phosphatidylglycerol tetraether,m/z1778; and diglycosyl phosphatidylinositol tetraether,m/z1866). The polar lipids ofMethanopyrus kandleriwere primarily glycolipids consisting of a series of archaeol lipids with one to six hexose units, composed primarily of mannose (mannose:glucose 9:1); phospholipids consisting of archaeol lipids (phosphatidylinositol diether; and a novel phosphatidylcholine diether,m/z802.7), and phosphoglycolipids as minor caldarchaeol lipids (primarily diglycosyl phosphatidylglycerol tetraether).Methanothermus fervidusextracts contained archaeol lipids (phosphatidylinositol diether; diglycosyl diether; and acetyldiglycosyl diether,m/z1016), and caldarchaeol lipids (glycosyl phosphatidylinositol tetraether,m/z1704; diglycosyl phosphatidylinositol tetraether; and acetyldiglycosyl phosphatidylinositol tetraether,m/z1907). Acetylation of a sugar residue occurred commonly in this thermophile and increased as cells entered the stationary growth phase. Lipid extracts ofSulfolobus acidocaldariuscontained detectable amounts of archaeol and hydroxyarchaeol analogs of phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine lipids, in addition to the dominant caldarchaeol lipids already reported. All four thermophiles contained both archaeol and caldarchaeol lipids and phosphoinositol head groups, but no single structural entity uniquely separated their lipids from those found previously in mesophilic archaea. By contrast, extremely halophilic archaea appear to be distinguished from the thermophilic archaea by the presence of a major phosphatidylglyceromethylphosphate lipid.Key words: ether lipids, mass spectrometry, hyperthermophiles, extreme halophiles, Archaea.