Phytochelatin synthases (PCSs) are activated by toxic metals/metalloids such as cadmium and arsenic and synthesize phytochelatins for detoxification of toxic elements. Rice (Oryza sativa L.) has two PCSs (OsPCS1 and OsPCS2), and we previously revealed that OsPCS1 has a higher responsiveness to arsenic than to cadmium, while OsPCS2 has a higher responsiveness to cadmium than to arsenic. Moreover, we found that the specific responsiveness of OsPCS1 to arsenic at rice nodes is a key factor in reducing arsenic in rice grains. However, the molecular characteristics of two PCSs in rice that contribute to the responsiveness to arsenic or heavy metals, including Cd, remain unclear. Here, we experimentally demonstrate that the C-terminal region in PCSs determines the responsiveness to arsenic or cadmium. We constructed chimeric proteins between OsPCS1 and OsPCS2 and performed an in vitro phytochelatin synthesis assay. A chimeric protein in which the 183 C-terminal amino acids of OsPCS2 were replaced with the 185 C-terminal amino acids of OsPCS1 showed higher responsiveness to arsenite than to cadmium, similar to OsPCS1. Contrary to expectations, mutations of cysteine residues that are unique to OsPCS1 or OsPCS2 had little influence on the responsiveness, although cysteine residues are reported to be representative of sites that interact with metals/metalloids. These results would enable the development of a breeding technology for reducing arsenic in rice grains by improving the arsenic-dependent activation of PCSs. (C) 2020 Elsevier Inc. All rights reserved.