Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that P. aerophilum requires the metal oxyanion WO42- for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 &mgr;M MoO42- did not replace WO42- for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 &mgr;M MoO42- to the culture medium. At lower tungstate concentrations (0.3 &mgr;M and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO42- concentrations (<0.7 &mgr;M). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO42- levels of 0.7 &mgr;M or higher. The high specific activity of the nitrate reductase enzyme observed at low WO42- levels (0.3 &mgr;M or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.