The effect of hydrogen peroxide (H2O2) of low concentration on thermotolerance of tall fescue (Festuca arundinacea cv. Barlexas) and perennial ryegrass (Lolium perenne cv. Accent) was studied following a foliar pretreatment with 10 mM H2O2. Antioxidative enzymes activities and antioxidant content were measured in both cool-season turfgrass cultivars under heat stress (38/30 °C, day/night) and control normal temperature (26/15 °C, day/night). While activities of catalase(CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione-dependent peroxidases (GPX) were enhanced by H2O2pretreatment during heat stress. APX, GR and GPX activities were significantly enhanced during heat stress. These were likely the most important antioxidative enzymes in tall fescue and perennial ryegrass protecting plants against heat stress. The thermotolerance was also concomitant with an increased glutathione pool, as evaluated by the significant increase of the total glutathione pool in two pretreated cultivars. The increase of POD, CAT, APX, GR activities and significant increase of GPX activity prior to the initiation of heat stress in pre-treatment plants suggested a possible role for H2O2as a signaling molecule protecting them against the subsequent heat-induced damage.