In order to make clear the response mechanism of antioxidant defense to heat stress at the subcellular level and hydrogen peroxideinduced heat tolerance of rhododendrons, we used three Rhododendron cultivars with different heat tolerance as experimental materials to analyze the effects of heat stress and H₂O₂ pretreatment on the level of reactive oxygen species (ROS) and the activities of antioxidant system in the chloroplasts, mitochondria and cytosol of Rhododendron leaves. The results showed that: (1) the order of the three Rhododendron cultivars according to their heat resistance were R. obtusum ‘Yanzhimi’, R. ‘Hongyue’ and R. ‘Hongshanhu’, respectively. (2) Under heat stress, the subcellular distribution of H₂O₂: cytosol > chloroplast > mitochondria. There was no significant difference in the production rate of superoxide radical (O₂^-·) in three subcellular organelles. The MDA content in cytosol was the highest in three Rhododendron cultivars, the subcellular distribution of MDA in R. ‘Hongyue’ and R. ‘Hongshanhu’ was the same with H₂O₂ distribution, but MDA in the mitochondria of R. obtusum ‘Yanzhimi’ was higher than that in the chloroplast. (3) Under heat stress, the order of superoxide dismutase (SOD) and peroxidase (POD) activities in R. obtusum ‘Yanzhimi’ and R. ‘Hongshanhu’: cytosol > chloroplast > mitochondria, however in R. ‘Hongyue’, the order was cytosol > mitochondria > chloroplast. The subcellular distribution of catalase (CAT) in R. obtusum ‘Yanzhimi’ was the same with R. ‘Hongyue’, their CAT activity in the mitochondria was higher than that in the cytosols and chloroplasts, but in R. ‘Hongshanhu’ the activity of CAT in the chloroplasts was the highest. (4) H₂O₂ pretreatment could improve the heat tolerance of rhododendron seedlings by enhancing the antioxidant defense ability of reducing the peroxidative damage under heat stress especially for the seedlings with poor heat tolerance. The results indicated that the subcellular distributions of ROS, MDA and antioxidant enzymes were different among three cultivars. The distribution of MDA was not completely consistent with that of ROS, and the chloroplasts of heatsensitive cultivars _R. ‘Hongyue’ and R. ‘Hongshanhu’ were more susceptible to oxidative damage than those of R. obtusum ‘Yanzhimi’. The effects of H₂O₂ pretreatment on the level of ROS and the activities of antioxidant enzymes in subcellular organelles appeared to be different among the three cultivars.