The Marselisborg WWTP (Aarhus, Denmark) fed the mainstream nitrification/denitrification tanks with excess sludge from a sidestream DEMON tank for more than three years to investigate if anammox can supplement conventional nitrification/denitrification in a mainstream of a temperate region. To evaluate this long-term attempt, anammox and also denitrification rates were measured in activated sludge from the main- and sidestream at 10, 20 and 30 C using 15N-labelling (stable isotope) experiments. The results show that anammox contributes by approximately 1% of the total nitrogen removal in the mainstream tanks and that anammox conversion rates there are approximately 800–900 times lower than in the DEMON. A distinct temperature dependence of both anammox and denitrification rates was also confirmed, however, results from different temperatures did not significantly alter relative shares, e.g. anammox rates in activated sludge from the nitrification/denitrification tanks are also negligible at 30 C. This indicates that the anammox bacteria abundance in the nitrification/denitrification tanks is too low to play an important role and that an adaptation to lower temperatures had not occurred. Additional in situ measurements in the nitrification/denitrification tanks further revealed that full nitrification dominates over partial nitritation. Dominant nitritation-anammox is therefore excluded per se and also nitrite shunt activities are not particularly supported.