This study investigated the cellular and subcellular compartmentation of Ni in the Eurasian serpentine species Alyssum murale, Alyssum bracteatum and Cleome heratensis and a non-serpentine population of A. murale (as a control) grown in hydroponic culture. Plant growth responses and Ni uptake clearly revealed the higher Ni tolerance of serpentine plants than the non-serpentine plants. Serpentine A. murale and A. bracteatum grew better at elevated (0.01 mM) Ni in the nutrient solution, supporting the view that the Ni hyperaccumulators have a higher requirement for Ni than normal plants. Low shoot Ni content of C. heratensis in response to the high Ni treatments indicated that this species employs an avoidance strategy for Ni tolerance. Energy-dispersive X-ray microanalysis showed that Ni was highly concentrated in the cell walls and cell lumen, most likely the vacuoles, of leaf epidermis of A. murale and A. bracteatum rather than in the mesophyll cells. EDX spectra from leaves of the non-serpentine A. murale suggested that Ni accumulated in both epidermal and mesophyll cells but not in the epidermal cell walls. Growth reduction and Ni toxicity in plants of the non-serpentine A. murale could be due to accumulation of Ni in the lumen of leaf mesophyll cells. Our data suggest that cellular and subcellular compartmentation are both possible mechanisms for Ni tolerance employed by the serpentine A. murale and A. bracteatum.