The effects of Cr(VI) stress (0, 75, 150 and 225 μM) on Cr accumulation, growth, d-aminolevulinic acid dehydratase (ALAD) activity, photosynthetic pigments content, proline content, malondialdehyde (MDA) content, antioxidant enzymes activity and 2-D protein profile were investigated using Cr-tolerant (Zeynelağa) and Cr-sensitive (Orza-96) barley cultivars. Zeynelağa accumulated significantly higher Cr in shoots than Orza- 96. Zeynelağa had a lower reduction in shoot dry weight, chlorophyll and carotenoid contents than Orza-96. Although Cr(VI) significantly reduced the ALAD activity, there was no consistent difference between ALAD activities of barley cultivars. Cr(VI) increased proline and MDA contents, but this effect was more pronounced in Orza-96. Cr(VI) stress caused an increase in the activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT). There was, in general, a marked difference between two barley cultivars in the extent of increased antioxidant enzyme activity. The results of the present study indicated that Zeynelağa seems to have better defense mechanisms to Cr(VI) stress compared to Orza-96. On the other hand, protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of 26 proteins.