Hydrogen evolution is observed in the green alga Scenedesmus obliquus after a phase of anaerobic adaptation. In this study we report the biochemical and genetical characterization of a new type of iron hydrogenase (HydA) in this photosynthetic organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The complete hydA cDNA of 2609 base pairs comprises an open reading frame encoding a polypeptide of 448 amino acids. The protein contains a short transit peptide that routes the nucleus encoded hydrogenase to the chloroplast. Antibodies raised against the iron hydrogenase from Chlamydomonas reinhardtii react with both the isolated and in Escherichia coli overexpressed protein of S. obliquus as shown by Western blotting. By analyzing 5 kilobases of the genomic DNA, the transcription initiation site and five introns within hydA were revealed. Northern experiments suggest that hydA transcription is induced during anaerobic incubation. Alignments of S. obliquus HydA with known iron hydrogenases and sequencing of the N terminus of the purified protein confirm that HydA belongs to the class of iron hydrogenases. The C terminus of the enzyme including the catalytic site (H cluster) reveals a high degree of identity to iron hydrogenases. However, the lack of additional Fe-S clusters in the N-terminal domain indicates a novel pathway of electron transfer. Inhibitor experiments show that the ferredoxin PetF functions as natural electron donor linking the enzyme to the photosynthetic electron transport chain. PetF probably binds to the hydrogenase through electrostatic interactions.