The impact of heat shock on minimising the activity of photosystem 2 (PS2) initiating high lipid peroxidation (POL) level and consequently changes in the enzymatic-antioxidant protective system was studied in seedlings of two Egyptian cultivars of barley (Giza 124 and 125). Heat doses (35 and 45 °C for 2, 4, 6, and 8 h) decreased chlorophyll (Chl) contents coupled with an increase in Chl a/b ratio, diminished Hill reaction activity, and quenched Chl a fluorescence emission spectra. These parameters reflect the disturbance of the structure, composition, and function of the photosynthetic apparatus as well as the activity of PS2. POL level, as dependent on the balance between pro- and anti-oxidant systems, was directly correlated with temperature, exposure time, and their interaction. Heat shock caused an increase in the electric conductivity of cell membrane, and malonyldialdehyde content (a peroxidation product) coupled with the disappearance of the polyunsaturated linolenic acid (C18:3), reflecting the peroxidation of membrane lipids which led to the loss of membrane selective permeability. Moreover, it induced distinct and significant changes in activities of antioxidant enzymes. Superoxide dismutase and peroxidase activities have been progressively enhanced by moderate and elevated heat doses, but the most elevated one (45 °C for 8 h) showed a decrease in activities of both enzymes. In contrast, catalase activity was reduced with all heat shocks. and F. El-Shintinawy ... [et al.].