Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K⁺ concentration, [K⁺]_cyt, and cytosolic pH, pH_cyt, in rice and wheat, plants with different tolerances to hypoxia. The [K⁺]_cyt and pH_cyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K⁺ from protoplasts of both plants after a lag-period of 300–450 s. The [K⁺]_cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K⁺ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K⁺]_cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pH_cyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K⁺ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.