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Climate changes such as heat waves often affect plant growth and pose a growing threat to natural and agricultural ecosystems. Elevated atmospheric CO2 can mitigate the negative effects of heat stress, but the underlying mechanisms remain largely unclear. We examined the interactive effects of elevated CO2 (eCO(2)) and temperature on the generation of the hydrogen peroxide (H2O2) and stomatal movement characteristics associated with heat tolerance in tomato seedlings grown under two CO2 concentrations (400 and 800 mu mol mol(-1)) and two temperatures (25 and 42 degrees C). eCO(2) ameliorated the negative effects of heat stress, which was accompanied by greater amounts of RESPIRATORY BURST OXIDASE 1 (RBOH1) transcripts, apoplastic H2O2 accumulation and decreased stomatal aperture. Silencing RBOH1 and SLOW-TYPE ANION CHANNEL, impeded eCO(2)-induced stomatal closure and compromised the eCO(2)-enhanced water use efficiency as well as the heat tolerance. Our findings suggest that RBOH1-dependent H2O2 accumulation was involved in the eCO(2)-induced stomatal closure, which participate in maintaining balance between water retention and heat loss under eCO(2) concentrations. This phenomenon may be a contributor to eCO(2)-induced heat tolerance in tomato, which will be critical for understanding how plants respond to both future climate extremes and changes in CO2.