Two extremely piezophilic bacteria DB21MT-2 and DB21MT-5 isolated from sediments from the Mariana Trench at 11,000 m were grown in the laboratory under low temperature (10oC) and high pressure (70 MPa) conditions. Phospholipid ester-linked fatty acids (PLFA) were analyzed by gas chromatography/mass spectrometry (GC/MS) and isotopic compositions of individual fatty acids were determined by gas chromatography isotope ratio mass spectrometry (GC-IRMS) in order to determine isotopic fractionation effects during biosynthesis. The piezophiles are characterized by fatty acids with carbon chains ranging from 14 – 22 carbons in length and by the presence of abundant polyunsaturated fatty acids (PUFA). Fatty acids from each culture exhibit a wide range of isotopic compositions (d13C vs. PDB). With two exceptions (C15:0 and C17:1 from DB21MT-2), all fatty acids are depleted in 13C relative to that of bulk carbon in the bacterial growth substrate. In addition, strain DB21MT-2 shows systematically higher (less negative) d13C values than strain DB21MT-5. The implications for isotope fractionation in fatty acid biosynthesis and the interpretation of isotopic composition of sedimentary fatty acids are discussed. It is suggested that the same type of microorganisms could have rather different d13C under the same growth conditions, and that sedimentary fatty acids with distinct d13C values do not necessarily have to originate from different organisms. Interpretation of d13C values of sedimentary biomarkers may be complicated by such large variations in d13C of fatty acids given that they are biosynthesized by the same type of microorganisms grown under identical conditions.