The impact of pretreatment with high temperature (45 degrees C for 45 min) on the UV-B tolerance of 4 barley cultivars (Hordeum vulgare L. 'Bulbul-89', 'Kalayci-97', 'Tarm-92', and 'Tokak-157/37') was examined. The response of the plants to treatment was evaluated by measuring the pigment content, chlorophyll a fluorescence, oxygen evolution, fraction of oxygen-evolving complex, proline content, UV-B-absorbing compounds (A(535) and A(300)), and stress markers (malondialdehyde, H2O2, and UV-B marker). Regardless of high temperature pretreatment, UV-B irradiation decreased the photosynthetic pigment content, photosystem II activity, oxygen evolution, and the fraction of oxygen-evolving complex in almost all of the barley cultivars. UV-B treatment significantly increased the proline content, UV-B-absorbing compounds, and stress markers. According to the findings, it can be deduced that short-term high temperature pretreatment might not provide a cross-tolerance to UV-B irradiation in the 4 barley cultivars studied; in fact, such exposure was found to aggravate the responses. In addition, although plants substantially accumulated the UV-B-absorbing compounds, the photosynthetic process might not be adequately protected from UV-B radiation.