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Digestion of bacterial biomass by three species of phagotrophic flagellates was studied using radioactive tracer techniques and short-term feeding experiments. Macromolecules of two different bacterial strains and natural limnic bacterioplankton were pulse-chase-labelled with one of the following precursors 3H-thymidine, 35S-/14C-methionine or 14C-leucine, before bacteria were fed to flagellates and radioactive labels were traced into flagellate macromolecules. The concentrations of prey and predators were monitored by flow cytometry. The aim of the work was to compare efficiencies of bacterial macromolecule accumulation by mixotrophic (Ochromonas) and heterotrophic (Spumella and Bodo) flagellates. We observed that flagellate accumulation efficiency of bacterial macromolecules labelled with thymidine (mean 15–30%, depending on flagellate species) was lower than of bacterial macromolecules labelled with amino acids (mean 26–68%). Heterotrophic flagellate species had similar accumulation efficiencies of bacterial molecules, when either leucine (26–42%) or methionine (31–41%) was used as a tracer. In contrast the mixotrophic flagellate accumulated significantly more residues of labelled methionine (68%) than of labelled leucine (54%). Methionine seems to be accumulated as an intact molecule and possibly Ochromonas preferentially accumulated methionine as an additional source of reduced sulphur. Protozoan accumulation efficiencies did not differ significantly whether the pulse-labelled bacterial prey were from growing or long-term starvation cultures. Our results suggest that labelled amino acids are more appropriate than labelled thymidine for studying transfer of bacterial biomass within food webs.