When methane (CH4) and O-2 are present, nitrogen can be removed from wastewater that does not contain other organic carbon sources. In this study, microbial activities during, methane-dependent denitrification (MDD) were investigated by adding inhibitors of methane-oxidation and denitrification. Sludge susceptible to MDD showed methane oxidation activity in the presence of CH4 and O-2, and denitrification activity with methanol and acetate under anoxic conditions. Methyl fluoride (CH3F) is known to inhibit methane oxidation. When CH3F was present, MDD did not occur, perhaps because methane oxidation was inhibited. Tungstate (WO42-), a known inhibitor of nitrate reduction, also lowered denitrification activity in the sludge, and partly inhibited methane oxidation. When WO42- was added to the medium, MDD almost ceased, perhaps because of a synergic inhibitory effect on denitrification and methane oxidation. These results show that both methane oxidation and denitrification contribute to MDD. (C) 2004 Elsevier Ltd. All rights reserved.