Here we provide the first direct evidence for the anammox process (anaerobic ammonium oxidation) in a lacustrine system, Lake Tanganyika, the second largest lake in the world. Incubations with 15N labelled nitrate showed that anammox occurred in the suboxic water layer at 100–110 m water depth. Anammox rates up to 10 nM N2 h−1 are comparable to those reported for the marine water column. Up to ∼13% of produced N2 could be attributed to the anammox process whereas the remainder was related to denitrification. Typical lipid biomarkers characteristic of anammox bacteria were found in filtered water from the depths where anammox occurred, thus supporting the presence of anammox bacteria. Further evidence is provided by fluorescence in situ hybridization (FISH), revealing up to 13 000 anammox bacteria cells per ml or 1.4% of all DAPI (4′-6-Diamidino-2-phenylindole)-stained cells. Phylogenetic analyses of partial 16S rRNA genes indicated the presence of sequences most closely related to the known anammox bacterium Candidatus‘Scalindua brodae’ (95.7% similarity). Using the incubation results, a total loss of 0.2 Tg N2 per year linked to anammox was estimated for the Northern basin of Lake Tanganyika.