In order to explore the physiological mechanism of paraquat resistance in tall fleabane, a widespread weed in Taiwan where resistance to this herbicide has been observed since 1980, the role of the antioxidative system was assessed. The susceptible (S) and resistant (R) biotypes of tall fleabane were distinguished clearly by the relative distribution frequency of injury index caused by 78 µM paraquat. Although malondialdehyde, an indicator for peroxidation damage to the plant, in the the R-biotype was not changed, in the S-biotype malondialdehyde increased within 2 h after treatment of 50 µM paraquat. Analysis of several antioxidants and pertinent enzymes revealed that ascorbate peroxidase activity was decreased by paraquat treatment in the S-biotype; and a lower basal level of ascorbate was present in the S-biotype as well. The maintenance of a high ratio of reduced glutathione to total glutathione, coupled with a pronounced and rapid increase of glutathione reductase (GR) activity in the the R-biotype, suggests that an active reduced glutathione/oxidized glutathione (GSH/GSSG) cycle is critical to paraquat resistance of tall fleabane. The decisive contribution of a functional GSH/GSSG cycle to paraquat resistance through an enhancement of GR activity in this weed was further confirmed by an experiment of exogenous application of ascorbate.