The conditions for production of nitrous oxide from the process of denitrification are studied by using activated sludge precultured with batch system. The activated sludge was obtained in the subsequent operations. The production of nitrous oxide was not observed in case of the sludge precultured with carbon rich substrate in spite of the change of pH and/or load of organic carbon. On the other hand, in the case of sludge precultured with TOC of less than 0.028 kg/kg-MLSS . d, nitrous oxide was produced when of pH was less than 7.0. The phenomena are recognized regardless of load of organic carbon.