We cloned a bacterial copper-containing nitrite reductase (NirK) homolog gene of Aspergillus oryzae (AonirK). Alignment showed that amino acid residues crucial for copper binding are conserved in the deduced sequence of the fungal protein. The recombinant protein exhibited distinct nitrite reductase activity, and its absorption and EPR spectra showed the presence of type 1 and type 2 copper atoms in the molecule. AonirK transcriptionally responded to denitrification conditions. Although the denitrifying activity of A. oryzae was weak under the conditions employed, high expression of the gene in the fungal cells enhanced the denitrifying activity 6-fold, accompanied by distinct cell growth. Furthermore, the highly expressed AoNirK was subcellularly localized to the mitochondria. The results demonstrated that AoNirK is responsible for fungal denitrification. Discussion is added on the novel insight concerning the origin and evolution of the mitochondrion provided by the findings for eukaryotic NirKs.