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Prokaryotic activities contribute to geochemical cycles on an Earth environment. To investigate roles of the prokaryotes in the environments, carbon and hydrogen isotopic compositions of lipid biomarkers can be a useful tool. This review describes carbon and hydrogen isotope fractionation associated with variety of metabolic and lipid synthetic pathways, factors controlling the isotope fractionation, and their applications into natural environments. Isotopic compositions of prokaryotic biomarkers potentially provide much information such as source organisms and environmental factors when they were biosynthesized.