Abstract Two classes of procaryotic organisms were cultured on specific inorganic and organic nitrogenous substrates. The organisms fractionated these substrates in characteristic ways during the synthesis of their cellular biochemical compounds. Blue-green algae, Anabaena sp., were raised on molecular nitrogen, nitrate and ammonia in the presence of excess carbon dioxide. The difference between δ 15N of either nitrate or ammonium and the δ 15N of the algae grown on either source was 13%o. The δ 15N of Anabaena that fixed N 2 was 2% lighter than the nitrogen gas supplied to the algae. These fractionations are associated with enzymatic incorporation of the nitrogen into the cell. A heterotrophic bacterium, Vibrio harveyi was grown on a single amino acid as its source of both nitrogen and carbon. Cells grown on glutamic acid were enriched in 15N relative to substrate, whereas those grown on alanine were depleted in 15N compared to source nitrogen. The bacterial cultures were enriched in 13C relative to the substrate. These cultures were then hydrolyzed and individual amino acids isolated and isotopically analyzed. The isotopic compositions of the amino acids have a wide range of values; most appear to have isotope fractionations associated with the metabolic pathways in their synthesis. These results and the application of the coupled separation-isotopic analysis of amino acids yield a better understanding of comparative biochemistry for these organisms. Such analyses offer valuable information for the tracing of biosynthesis and early diagenesis to help explain the fossil record.