Objectives To synthesize hydrazine (N2H4) from ammonium and hydroxylamine (NH2OH) using an anaerobic ammonium oxidation (anammox) bacterium, Candidatus Kuenenia stuttgartiensis. Results K. stuttgartiensis cells were anoxically cultivated with the addition of ammonium (2 mM) and NH2OH (1-100 mM) at pH 6-10.5, and 4-65 degrees C to examine the favorable cultivation conditions for N2H4 production. The influence of NH2OH concentration was more prominent than that of pH and temperature, and NH2OH concentration higher than 1 mM deteriorated N2H4 yields significantly. The following conditions were found to be favorable for N2H4 production using K. stuttgartiensis cells: pH 9, 38 degrees C, and < 1 mM NH2OH. In a continuous-feed system operated at these conditions, K. stuttgartiensis cells produced N2H4 with a maximum concentration of 0.65 mM, which is the highest N2H4 concentration previously reported in biological processes. Conclusions Optimal cultivation conditions for K. stuttgartiensis for N2H4 production were successfully determined, and the present study is the first to document potential biological N2H4 production using anammox bacteria.