Summary: The gene homologous to glutathione synthetase of was inactivated in the cyanobacterium sp. PCC 7942. The region of genomic DNA including the mutation site was isolated from the mutant by plasmid rescue and the native gene of the wild-type was cloned from a genomic DNA library of the wild-type using the flanking DNA as a probe. The wild-type gene, designated , encodes a polypeptide of 323 amino acids with a molecular mass of 35 kDa. The deduced amino acid sequence resembles glutathione synthetases of bacteria, but not those of higher organisms. When was overexpressed in , glutathione synthetase activity was increased markedly in the extract. In addition, the sp. PCC 7942 mutants had lost their ability to synthesize glutathione. These findings demonstrate that the gene of sp. PCC 7942 is a structural gene for glutathione synthetase and is involved in the biosynthesis of glutathione.