The soluble hydrogenase I (H 2:NADP + oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P. furiosus hydrogenase I preparation (PF H 2ase I) has been used as biocatalyst in the enzymatic production and regeneration of β-1,4-nicotinamide adenindinucleotide phosphate, reduced form (NADPH), utilizing cheap molecular hydrogen and forming protons as the only side-product. Any excess of dihydrogen can be removed easily. It could be demonstrated, that this hyperthermophilic hydrogenase exhibits a high stability under reaction conditions. Generation as well as regeneration of NADPH were performed in batch and repetitive batch experiments with recyclisation of the biocatalyst. In two repetitive batch-series 6.2 g l −1 NADPH could be produced with a total turnover number (ttn: mol produced NADPH/mol consumed enzyme) of 10,000. Utilizing the thermophilic NADPH-dependent alcohol dehydrogenase from Thermoanaerobium spec. (ADH M) coupled to the PF H 2ase I in situ NADPH-regenerating system, two prochiral model substrates, acetophenone and (2 S)-hydroxy-1-phenyl-propanone (HPP), were quantitatively reduced to the corresponding ( S)-alcohol and (1 R,2 S)-diol. An e.e. >99.5% and d.e. >98%, respectively, with total turnover numbers (ttn: mol product/mol consumed cofactor NADP +) of 100 and 160 could be reached.