RATIONALE: Studies of archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in the environment and cultures have exclusively focused on compounds with fully saturated alkyl moieties. Here we report a number of novel unsaturated GDGTs (uns-GDGTs) whose alkyl chains contain up to six double bonds and zero to two cyclopentyl moieties. METHODS: The identification of these lipids was achieved via comparison of lipid distribution before and after hydrogenation, characteristic retention time patterns, and diagnostic ions using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and ether cleavage products using gas chromatography/mass spectrometry (GC/MS). Isomerism resulting from different unsaturation patterns in the alkyl moieties was observed and specific positions of double bonds in the biphytene and biphytadiene moieties were tentatively assigned. RESULTS: Uns-GDGTs were detected in sediment and microbial mat samples as both core lipids (CLs) and intact polar lipids (IPLs) associated with mono- or diglycosyl or phosphatidylglycerol headgroups. However, these lipids were overlooked in past investigations because conventional methods for archaeal lipid analysis are unsuitable for uns-GDGTs. Samples from distinct marine environments (Black Sea, Cariaco Basin, Discovery Basin, Eastern Mediterranean Sea, upwelling area off NW Africa, and seep sites off Crimea and Pakistan) were screened for uns-GDGTs using a new LC/MS protocol. The results show that uns-GDGTs contribute significantly to the archaeal lipid pool in anoxic methane-rich environments (Black Sea, Cariaco Basin, and both seep sites) but they were barely detected in the oxic or hypersaline settings. CONCLUSIONS: The characteristic distribution of uns-GDGTs implies that they are attractive targets for future studies aiming at the chemotaxonomy of uncultivated archaea and regulation of uns-GDGT biosynthesis.