Genes encoding phytochelatin (PC) synthase have been found in higher plants, fission yeast and worm. Recently, kinetic and mutagenic analyses of recombinant PC synthase have been revealing the molecular mechanisms underlying PC synthesis, however, a conclusive model has not been established. To clarify the mechanism of PC synthase found in eukaryotes, we have compared the two-step reactions catalyzed by the prokaryotic Nostoc PC synthase (NsPCS) and the eukaryotic Arabidopsis PC synthase (AtPCS1). Comparative analysis shows that in the first step of PC synthesis corresponding to the cleavage of gamma-glutamylcysteine (gamma-EC) from glutathione (GSH), free GSH or PCs acts as a donor molecule to supply a gamma-EC unit for elongation of the PC chain, and heavy metal ions are required to carry out the cleavage. Furthermore, functional analyses of various mutants of NsPCS and AtPCS1, selected by comparing the sequences of NsPCS and AtPCS1, indicate that the N-terminal region (residues 1-221) in AtPCS1 is the catalytic domain, and in this region, the Cys(56) residue is associated with the PC synthesis reaction. These results enable us to propose an advanced model of PC synthesis, describing substrate specificity, heavy metal requirement, and the active site in the enzyme.