Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH•), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper-transport activity: Col-0, high-affinity copper transporter COPT1-overexpressing (C1OE) seedlings, and T-DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm) stimulated a dose-dependent Gd3+ and verapamil sensitive net Ca2+ influx in the root apex but not in mature zone. C1OE also showed a fivefold higher Cu-induced K+ efflux at the root tip level compared with Col-0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1OE seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd3+. Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH•-sensitive Ca2+ and K+ transport systems.