Non-photochemical chlorophyll fluorescence quenching (NPQ) plays a major role in the protection of the photosynthetic apparatus against damage by excess light, which is closely linked to the production of reactive oxygen species (ROS). The effect of a short heat treatment on NPQ and ROS production was studied with detached tobacco leaves by fluorescence imaging of chlorophyll and of the ROS sensor dye HO-1889NH. NPQ was stimulated >3-fold by 3 min pre-treatment at 44°C, in parallel with suppression of CO2 uptake, while no ROS formation could be detected. In contrast, after 3 min pre-treatment at 46°C, NPQ was suppressed and ROS formation was indicated by quenching of HO-1889NH fluorescence. After 3 min pre-treatment at 46°C and above, partial inactivation of ascorbate peroxidase and light-driven accumulation of H2O2 was also observed. These data are discussed as evidence for a decisive role of the Mehler ascorbate peroxidase or water–water cycle in the formation of the NPQ that reflects down-regulation of PSII.