The effect of heavy metals on plasma membrane (PM) H+-ATPase (EC 188.8.131.52) activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H+-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 μM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H+-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H+-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H+-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H+-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H2O2 in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H+-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H+-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants.