The seedling-lethal nuclear Arabidopsis hcf101 (high chlorophyll fluorescence) mutant is impaired in photosynthesis and complemented by the wild-type HCF101 cDNA. Photosystem I (PSI) activity is abolished, and PSI core complexes fail to accumulate in hcf101, whereas levels of other thylakoid membrane proteins are unaffected. Northern and in vivo labelling analyses as well as studies on polysome loading show that PSI transcript levels and translation rates of proteins, which belong to PSI, are normal in hcf101. PSI-specific fluorescence at 77 K is shifted from 735 to 728 nm in hcf101, indicating that exitons cannot efficiently be transferred to the PSI reaction centre, whereby the PSI antenna is almost unaffected. Mutant plants not only fail to accumulate mature PSI, which contains three [4Fe-4S]clusters (FSCs), but also are characterized by reduced levels of the soluble FSC-containing complex ferredoxin-thioredoxin reductase (FTR) in the stroma. Inhibited FTR maturation is not a secondary effect stemming from lack of PSI because the mutant hcf145, which also lacks PSI, accumulates FTR at normal levels. Levels of the [2Fe-2S] cluster-containing soluble and membrane proteins, ferredoxin and PetC, respectively, were unchanged in hcf101 plants. These data suggest a specific role of HCF101 in FSC biogenesis. HCF101 is plastid localized and belongs to an ancient and universally conserved family of P-loop ATPases previously designated as the 'MRP' (metGrelated protein) family. The function identified for HCF101 suggests a new designation, FSC, for this family.