Synthesis of phytochelatins (PCs), heavy-metal-sequestering peptides, in the marine green alga, Dunaliella tertiolecta, was evaluated under various conditions of exposure to heavy metals. To investigate the effect of heavy metals on both PC synthesis and their upstream biosynthetic reactions, an ion-pair-HPLC system was developed in this study, by which PCs and their biosynthetic intermediates, cysteine (Cys), gamma -glutamylcysteine (gamma EC) and glutathione (GSH), could be determined simultaneously with high sensitivity. When the cells were exposed to Zn2+, the level of PCs was maximal at 200 muM and significantly higher than that obtained after exposure to 400 muM Cd2+, which is the strongest inducer of PC synthesis in higher plants in vivo and in vitro as well as in microalgae. The predominant PC subtype was PC4, followed by PC3 and PC5, whereas PC2, which is generally abundant in higher plants, has the lowest level among PC2 to PC5. These results suggest that the characteristics of PC synthase in D. tertiolecta including the requirement of heavy metals for its catalysis and substrate specificity towards GSH and PCn are considerably different from those in higher plants and other algae. While PC synthesis proceeded in the heavy-metal-treated cells, the level of GSH did not appreciably change. To maintain the same size of the GSH pool, GSH must be newly synthesized to balance the amount consumed for PC synthesis.