To identify genes necessary for the photoproduction of H(2) in Chlamydomonas reinhardtii, random insertional mutants were screened for clones unable to produce H(2). One of the identified mutants, denoted hydEF-1, is incapable of assembling an active [Fe] hydrogenase. Although the hydEF-1 mutant transcribes both hydrogenase genes and accumulates full-length hydrogenase protein, H(2) production activity is not observed. The HydEF protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase. In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. All organisms with [Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG, which, prior to this study, were of unknown function. Within several prokaryotic genomes HydE, HydF, and HydG are found in putative operons with [Fe] hydrogenase structural genes. Both HydE and HydG belong to the emerging radical S-adenosylmethionine (commonly designated "Radical SAM") superfamily of proteins. We demonstrate here that HydEF and HydG function in the assembly of [Fe] hydrogenase. Northern blot analysis indicates that mRNA transcripts for both the HydEF gene and the HydG gene are anaerobically induced concomitantly with the two C. reinhardtii [Fe] hydrogenase genes, HydA1 and HydA2. Complementation of the bx;1C. reinhardtii hydEF-1 mutant with genomic DNA corresponding to a functional copy of the HydEF gene restores hydrogenase activity. Moreover, co-expression of the C. reinhardtii HydEF, HydG, and HydA1 genes in Escherichia coli results in the formation of an active HydA1 enzyme. This represents the first report on the nature of the accessory genes required for the maturation of an active [Fe] hydrogenase.