Fe-hydrogenase-specific degenerate primers were used in polymerase chain reactions with single-stranded Scenedesmus obliquus cDNA as template. A resulting 435-bp fragment was used to screen a cDNA library from S. obliquus. A 1.6-kb cDNA clone, containing the 3'-end of an open reading frame, was obtained. Its deduced amino acid sequence contains the conserved H-cluster motives unique for Fe-hydrogenases. Any other FeS-clusters seem to be absent. Phylogenetically, the enzyme is closely related to the Trichomonas vaginalis Fe-hydrogenase. Northern blot analysis shows that the Fe-hydrogenase is constitutively expressed in S. obliquus. Southern blot analysis of plastidic, mitochondrial, and nuclear DNA reveals that the enzyme is a single-copy gene encoded in the nucleus. Effects of transcriptional and translational inhibitors on H2-metabolism reveal the involvement of a chloroplast-encoded protein.