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The activity of dye-linkedd-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicumJCM 9189. The dye-linked d-proline dehydrogenase was a membrane associated enzyme and was solubilized from the membrane fractions by treatment with Tween 20. The solubilized enzyme was purified 34-fold in the presence of 0.1% Tween 20 by four sequential chromatographies. The enzyme has a molecular mass of about 145 kDa and consisted of homotetrameric subunits with a molecular mass of about 42 kDa. The N-terminal amino acid sequence of the subunit was MKVAIVGGGIIGLFTAYHLRQQGADVVI. The enzyme retained its full activity both after incubation at 80 °C for 10 min and after incubation in the range of pH 4.0–10.0 at 50 °C for 10 min. The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, andd-proline was the most preferred substrate among thed-amino acids. The Michaelis constants ford-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively. Δ1-Pyrroline-2-carboxylate was identified as the reaction product from d-proline by thin layer chromatography. The prosthetic group of the enzyme was identified to be FAD by high-performance liquid chromatography. The gene encoding the enzyme was cloned and expressed in Escherichia coli.The nucleotide sequence of the dye-linked d-proline dehydrogenase gene was determined and encoded a peptide of 363 amino acids with a calculated molecular weight of 40,341. The amino acid sequence of the Pb. islandicumenzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those ofd-alanine dehydrogenases from the mesophiles so far reported. This shows that the membrane-bound d-proline dehydrogenase from Pb. islandicumis a novel FAD-dependent amino acid dehydrogenase.