A protocol has been developed for the preparation of isotopically characterized internal standards for compound-specific isotope analysis. Fatty acids and their corresponding methyl esters are loaded into flame seal vials by a protocol carefully designed to minimize isotopic fractionation and contamination. Carbon isotope analysis of randomly selected vials yields isotopic precision with an average standard deviation of 0.09 parts per thousand (delta(13)C(PDB)). Absolute values for carbon isotope ratios are determined against an international standard (NIST RM8541: USGS24). The procedure is general and may be used for all compounds soluble in volatile solvents. The fatty acid and fatty acid methyl esters standards developed here will be useful for day-to-day quality control and assessment of derivative carbon addition due to methylation of fatty acids.