Vacuolar H -ATPase (V-ATPase; EC 3.6.3.14) is the main enzyme responsible for generating a proton gradient across the tonoplast. Under cadmium (Cd) stress conditions, V-ATPase activity is inhibited. In the present work, hydrogen sulfide (H S) and hydrogen peroxide (H O ) cross-talk was analyzed in cucumber (Cucumis sativus L.) seedlings exposed to Cd to explain the role of both signaling molecules in the control of V-ATPase. V-ATPase activity and gene expression as well as H S and H O content and endogenous production were determined in roots of plants treated with 100 μM CdCl and different inhibitors or scavengers. It was found that H S donor improved photosynthetic parameters in Cd-stressed cucumber seedlings. Cd-induced stimulation of H S level was correlated with the increased activities of the H S-generating desulfhydrases. Increased H O and lowered H S contents in roots were able to reduce V-ATPase activities similar to Cd. H O and H S-induced modulations in V-ATPase activities were not closely related to the transcript level of encoding genes, suggesting posttranslational modifications of enzyme protein. On the other hand, exogenous H O raised H S content in root tissues independently from the desulfhydrase activity. Although treatment of control plants with H S significantly stimulated NADPH oxidase activity and gene expression, H S did not affect H O accumulation in roots exposed to Cd. The results suggest the existence of two pathways of H S generation in Cd-stressed cucumber roots. One involves desulfhydrase activity, as was previously demonstrated in different plant species. The other, the desulfhydrase-independent pathway induced by H O /NADPH oxidase, may protect V-ATPase from inhibition by Cd.