Ultraviolet B (UV-B) irradiation has an adverse effect on plant cells because it causes the generation of reactive oxygen species (ROS). This is scavenged by some endogenous defense systems. In this study, the activities of H2O2-detoxifying antioxidant enzymes in 4 barley cultivars pretreated with high temperature or NaCl before UV-B irradiation were investigated. The stress response in the seedlings was determined by measuring the photosynthetic performance index (PI), which significantly decreased in all barley cultivars under almost all treatments compared to their controls. Activities of 3 H2O2-detoxifying antioxidant enzymes (ascorbate peroxidase, glutathione reductase, and peroxidase) significantly increased as a result of all treatments. The results may suggest that UV-B irradiation induces major H2O2-detoxifying enzyme activities, and pretreatments, especially with 200 mM of NaCl, enhance the response of enzyme activities in these 4 barley cultivars. Changes in the PI responses could indicate that the induction of antioxidant enzymes might not be enough to protect from oxidative stress induced by UV-B radiation in the barley cultivars investigated in the study.