The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles, but relative rates of accumulation/association of metals with vesicles were Ca > Cd >> Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in oat roots.