The isotope fractionation of carbon from substrates possessing different isotope ratios into fatty acids of polar lipids and amino acids was determined for four different fungi (Rhizopus arrhizus, Mortierella isabellina, Fusarium solani, Aspergillus niger). Carbon isotope ratios of fungi closely followed that of the substrates. Palmitic acid (C16:0), derived from phospholipids, did not display a large carbon isotope fractionation against the substrate. Stearic acid (C18:0), however, was depleted in C-13 against C16:0 in all strains. The desaturation of C18:0 to oleic acid (C18:1omega9) had little effect on the carbon isotope ratio. The subsequent desaturation of C18:1omega9 to linolic acid (C18:2omega6,9) enriched the resulting C18:2omega6,9 by + 3.9% and varied little among strains. This result is important because C 18:2omega6,9 is often used as a biomarker in environmental studies. Most amino acids were enriched in C-13 compared to the substrates, but isoleucine and lysine were close to the isotope ratio of the substrate and phenylalanine and leucine were depleted. Interestingly, the carbon isotope ratios of many amino acids differed significantly among different species. A discriminant analysis based on the isotope ratio of four amino acids (Thr, Ile, Phe, Val) resolved the two phyla in the first discriminant function and all four strains in the first two discriminant functions and confirmed a taxon-specific manner of isotope fractionation. The derived rules provide the basis for the use of stable isotopes in environmental studies to elucidate the role of fungi in the carbon flow in the environment. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.