The fatty acid compositions of 5 freshwater heterotrophic flagellates were studied using laboratory cultures. The flagellates Bodo saltans, Paraphysomonas vestita, Spumella pudica, Polytoma uvella and Chilomonas paramecium were cultured on Synura medium enriched either with Régilait milk powder (MP medium; Régilait, St Martin Roche, France) or with Tabimin dry fish food (DFF medium; Tetra, Melle, France). The bacteria present in the culture media were also analyzed. The enrichment substrate was found to have a pronounced influence on the fatty acid profiles of the organisms. For example, flagellates grown on the MP medium were characterized by very low contents of polyunsaturated fatty acids (PUFA; 1.28 to 5.50% of total fatty acids), whereas those grown on DFF medium contained up to 13.20% PUFA. Irrespective of the species studied, the dominant PUFA were those of the (n-6) series. The presence of branched uneven-numbered fatty acids and of 18:1(n-7) within the protozoans reflects an intensive bacterivorous activity that is understandable in phagotrophic species such as B. saltans, P. vestita and S. pudica but is debatable in C. paramecium and P. uvella, as these species have been described as osmotrophic. The heterotrophic flagellates in this study seemed to have varying, but rather limited, capacities of PUFA biosynthesis and bioconversion that were partly related to the physicochemical conditions in the cultures (temperature and dissolved oxygen) and to the phylogenetic relations of the organisms. Species containing a leucoplast or a residual plastid reproduced in a very attenuated manner the characteristic PUFA profiles of autotrophic flagellates belonging to their taxonomic group. This was mainly the case for C. paramecium and P. uvella. Finally, our results suggest that in the natural environment, the PUFA contents of picoplanktonic prey and of dissolved organic matter are the main factors influencing the PUFA content of these flagellates.