Out of 1,343 mutant lines of rice mutated by sodium azide from the parental Japonica-type variety ‘Tainung 67’ (TNG67), a paraquat-susceptible line 1192 and a paraquat-tolerant line 72 were selected using whole seedlings at the four-leaf stage and leaf segments at the tillering stage as test materials. Further selection from progenies of these two mutant lines yielded the susceptible 1192-11 (S) and tolerant 72-16 (T), which were studied herein. Chlorophyll fluorescence, electrolyte leakage, and lipid peroxidation were measured for leaf segments of rice following treatment with 0.1 mM paraquat. A comparison of these responses among the three rice lines (TNG67, 72-16, and 1192-11), revealed a higher tolerance to paraquat in the tolerant mutant line 72-16 and the parental variety TNG67 than in the susceptible mutant 1192-11. Analysis of the antioxidative system in paraquat-treated leaf segments showed that the reduced form of glutathione (GSH) and the ratio of GSH to total glutathione increased by 3.5-fold within 6 h after treatment (HAT) and up to 5-fold 9 HAT in the T line, as compared with the S line. In view of the high activities of both dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in paraquat-treated leaves of TNG67 and the T line, the antioxidative effect of the ascorbate–glutathione cycle is hereby proposed to play an essential role in paraquat tolerance of rice. Pretreatment of rice segments with spermine enhanced DHAR and GR activities as well as paraquat tolerance of the S line. These results suggest that the activity of ascorbate–glutathione cycle induced by spermine is involved in rice tolerance to this herbicide. Although kinetics studies showed no significant difference among the three rice lines in paraquat inhibition of GR, a lower affinity of enzyme to substrate (Km) in TNG67 and the T line and a higher maximal reaction rate (Vmax) in the T line for the oxidized glutathione substrate (GSSG) were detected. These observations further implicate the importance of glutathione reductase in paraquat tolerance of rice