Lipids from prokaryotic cell membranes can serve as sources of information on the biogeochemistry and microbial ecology of natural ecosystems. Traditionally, apolar derivatives of the intact polar membrane molecules, e.g., fatty acids, have been the major target of lipid-based biogeochemical studies. However, when still intact, i.e., as glycerol esters and ethers with attached polar headgroups, membrane lipids are diagnostic for living prokaryotes, which makes them excellent biomarkers for the study of in situ microbial processes in geological systems such as sediments or soils. Intact polar lipids (IPLs) are attractive analytical targets because they are taxonomically more specific than their apolar derivatives and avoid exclusion of signals from prokaryotes that primarily build their membranes with ether-bound lipids such as archaea and some bacteria. Here we report results from analyses of IPLs in pure cultures of biogeochemically relevant prokaryotes and marine sediments by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-IT-MSn). This technique is suitable for screening of biomass and environmental samples for distinctive taxonomic structural features such as distribution of polar lipid headgroups, types of bonds between alkyl moiety and glycerol backbone, and the chain length and degree of unsaturation in the alkyl moieties. We present analytical protocols to decipher structural information from mass spectral data. The IPL contents in selected archaeal and bacterial species are diverse and qualify as molecular fingerprints. Applied to marine sediments, the approach provided detailed information on the dominant microbial groups. The IPLs from bacterial members of anaerobic methanotrophic communities in surface sediments at Hydrate Ridge resemble those found in Desulfosarcina variabilis. The presence of dietherglycerophospholipids, however, suggests the presence of other bacteria possibly affiliated with the deepest phylogenetic branches in the tree of life. Sediments from ∼90 m below the seafloor on the Peruvian continental margin are dominated by intact archaeal tetraethers with glycosidically bound hexoses as headgroups, consistent with a significant fraction of the community being archaea. Additional calditol-based tetraethers imply that the sedimentary archaea are taxonomically linked to the crenarchaeal Sulfolobales. Copyright © 2004 John Wiley & Sons, Ltd.