Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h) GSH in biological nitrogen fixation (BNF). The (h) GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the gamma-glutamylcysteine synthetase (gamma ECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h) GSH content was correlated with a reduction in the nodule size. Conversely, gamma ECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h) GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.