Among cyanobacteria, the heterocystous, N2-fixing Anabaena variabilis and the unicellular Anacystis nidulans have recently been shown to possess an NAD+-dependent, bidirectional hydrogenase. A 5.0 kb DNA segment of the A. nidulans genome is now identified to harbor the structural genes hoxUYH coding for three subunits of the bidirectional hydrogenase. The gene arrangement in A. nidulans and in A. variabilis is remarkably dissimilar. In A. nidulans, but not in A. variabilis, the four accessory genes hoxW, hypA, hypB and hypF could be identified downstream of hoxH. An insertional homozygous mutant in hoxH from A. nidulans was completely inactive in performing Na2S204-dependent H2 evolution but could utilize the gas with almost 50% of the activity of the wild type. These findings with the first defined hydrogenase mutant in any photosynthetic, 02-evolving microorganism indicate that the unicellular cyanobacterium A. nidulans possesses both an uptake and a bidirectional hydrogenase. The physiological role(s) of the two hydrogenases in unicellular non-N2-fixing cyanobacteria is not yet understood.