The thiol tripeptides glutathione (GSH) and homoglutathione (hGSH) are very abundant in legume root nodules and their synthesis is catalyzed by the enzymes -glutamylcysteine synthetase (ECS), GSH synthetase (GSHS), and hGSH synthetase (hGSHS). As an essential step to elucidate the role of thiols in N2 fixation we have isolated cDNAs encoding the three enzymes and have quantified the transcripts in nodules. Assay of enzyme activities in highly purified nodule organelles revealed that ECS is localized in the plastids, hGSHS in the cytosol, and GSHS in the cytosol and mitochondria. These results are consistent with sequence analyses. Subcellular fractionation of nodules also showed that bacteroids contain high thiol concentrations and high specific ECS and GSHS activities. Results emphasize the role of nodule plastids in antioxidant protection and in control of thiol synthesis, and suggest that plastids may be important in the stress response of nodules. Overall, our results provide further evidence that thiol synthesis is critical for nodule functioning.This work was supported by the Comisión Interministerial de Ciencia y Tecnología and the European Commission (grant nos. 2FD97-1101 and PB98-0522 to M.B.), by the Dirección General de Enseñanza Superior e Investigación Científica and the British Council (Acción Hispano-Británica HB98-163 to M.B. and N.J.B.), by a Marie Curie grant from the European Commission (to I.I.-O.), and by the Biotechnology and Biological Sciences Research Council (to N.J.B.). J.F.M., M.A.M., M.C.R., and M.R.C. were the recipients, respectively, of a postdoctoral contract from the Ministry of Education and Culture (Spain), a predoctoral fellowship from the Gobierno Vasco, and two predoctoral fellowships from the Ministry of Education and Culture.Peer reviewed