© 2014 John Wiley & Sons Ltd. Oxygen deprivation is a key determinant of root growth and functioning under waterlogging. In this work, changes in net K+ flux and membrane potential (MP) of root cells were measured from elongation and mature zones of two barley varieties under hypoxia and anoxia conditions in the medium, and as influenced by ability to transport O2 from the shoot. We show that O2 deprivation results in an immediate K+ loss from roots, in a tissue- and time-specific manner, affecting root K+ homeostasis. Both anoxia and hypoxia induced transient membrane depolarization; the extent of this depolarization varied depending on severity of O2 stress and was less pronounced in a waterlogging-tolerant variety. Intact roots of barley were capable of maintaining H+-pumping activity under hypoxic conditions while disrupting O2 transport from shoot to root resulted in more pronounced membrane depolarization under O2-limited conditions and in anoxia a rapid loss of the cell viability. It is concluded that the ability of root cells to maintain MP and cytosolic K+ homeostasis is central to plant performance under waterlogging, and efficient O2 transport from the shoot may enable operation of the plasma membrane H+-ATPase in roots even under conditions of severe O2 limitation in the soil solution.