The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2O2) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells.