Pteris vittata (Chinese brake fern) has potential for phytoremediation of As-contaminated sites. In this study, the synthesis of total thiols and acid-soluble thiols in P. vittata was investigated under arsenic exposure. The strong and positive correlation between As concentration and acid-soluble thiols in plant leaflets suggests that acid-soluble thiols may play a role in As detoxification. A major As-induced thiol was purified and characterized. A molecular ion (M + 1) of 540 m/z suggests that the thiol was a phytochelatin (PC) with two base units (PC(2)). However, the ratios of acid-soluble thiols to As in leaflets exposed to As ranged from 0.012 to 0.026, suggesting that only a very small part of As is complexed by PC(2). PCs could play a minor detoxification role in this hyperaccumulator. A PC-independent mechanism appears to be mainly involved in As tolerance, while PC-dependent detoxification seems to be a supplement.