A gene (PH0311) encoding a hypothetical protein from the genome sequence data of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and over-expressed in Escherichia coli. The purified recombinant protein was found to possess FAD-dependent NADH oxidase activity, although it lacked sequence homology to any other known general NADH oxidase family. The product of the PH0311 gene was thus designated PhNOX (NADH oxidase from Pyrococcus horikoshii), with an estimated molecular weight of 84 kDa by gel filtration and 22 kDa by SDS-PAGE, indicating it to be a homotetramer of 22 kDa subunits. PhNOX catalyzed the oxidation of reduced ${eta}$-NADH with subsequent formation of $H_2O_2$ in the presence of FAD as a cofactor, but not ${alpha}$-NADH, ${alpha}$-NADPH, or ${eta}$-NADPH. PhNOX showed high affinity for ${eta}$-NADH with a Km value of 3.70 ${mu}$M and exhibited optimum activity at pH 8.0 and 95$^{circ}C$ as it is highly stable against high temperature.