An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD(+)-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP(+)-dependent protein from Desulfovibrio fructosovorans. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A. eutrophus enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY, respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 7119 and Anacystis nidulans was amplified by PCR. Using the labeled segment from A. 7119 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 7119, Anabaena cylindrica, Anacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A. variabilis. Therefore the 8.9-kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-dependent H2 evolution corroborating the molecular biological demonstration of the NAD(P)(+)-dependent hydrogenase in cyanobacteria.