Compartmentation of metals in specific tissues, cells, and subcellular compartments is considered a metal-tolerance mechanism in metal hyperaccumulator plants. In this study, we investigated the accumulation of Ni in the trichomes of a serpentine endemic Ni hyperaccumulator plant, Alyssum inflatum, native to western Iran. Elemental analysis of plants from their natural habitat showed that the Ni concentration of trichomes was not higher than in the shoot, suggesting that Ni does not preferentially accumulate in trichomes. Treatment of plants by adding different concentrations of Ni to the growth medium showed that staining of trichomes with dimethylglyoxime (a specific stain for Ni) increased as concentrations of external Ni increased. Accumulation occurred in the base of trichomes and, by increasing the concentration of Ni, accumulation extended to the rays and cell walls. The results showed that trichomes can accumulate high concentrations of Ni and that Ni accumulation can be under the control of Ni concentration in the shoot.