Aluminium (Al) tolerance in wheat (Triticum aestivum L.) is associated with the Al-activated efflux of malate and K+ from the root apices. We tested the hypothesis that these Al-activated ion fluxes would induce changes in the membrane potential (Vm) and that these responses would differ between wheat genotypes that differ in Al tolerance. Within minutes of exposing wheat roots to 50 μm AlCl3, a significant depolarisation was measured in the Al-tolerant ET8 genotype but not in a near-isogenic, Al-sensitive genotype, ES8. We investigated the ion movements that may be responsible for these changes in Vm by measuring real-time fluxes of Cl-, H+ and K+ at the root apices of wheat seedlings using the non-invasive microelectrode ion flux estimation (MIFE) technique. Addition of 50 μm AlCl3 to the bathing solution stimulated an increase in K+ efflux and H+ influx in ET8 but not in ES8. The differences between the genotypes were sustained for 24 h and were observed only at the elongating zone and not the meristematic zone. After 24 h Al increased Cl- influx in ET8 but inhibited ES8 influx in a dose-dependent manner. These results provide new temporal and spatial information on the Al-activated ion fluxes from intact wheat plants.