The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.