Glutathione (GSH) is considered to be important components involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to characterise the nucleotide sequence of genes involved in glutathione biosynthesis (ci-gclc, ci-gclm and ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the expression of these genes after in vivo exposure to Cd, Cu and Zn. These genes exhibit a good level of sequence conservation with corresponding metazoan orthologous, especially for residues important for the enzyme activities. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM resulting as sister group of vertebrate GCLMs. Our data highlighted a statistically significant increase in gene expression after metal treatments. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the expression of these genes may directly depend on metals and/or reactive oxygen species. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. inestinalis to survive in the continued elevated presence of heavy metals in the environment.