The subcellular distribution of arsenic (As) in Pteris vittata L., an As-hyperaccumulator, was studied to determine As compartmentalization and to explore the mechanisms that confer As tolerance. When the plant was grown in a nutrient solution without additional As, most of the accumulated As was isolated to the cell wall. However, in plants growing in a nutrient solution containing 0.1 or 0.2 mmol/L As, approximately 78% of the total As accumulated within the pinna. The proportions of As accumulation in the cytoplasmic supernatant fraction were 78% of that in the pinna and 61% of that in the plant. In either treatment group (0.1 or 0.2 mmol/L As), the fraction containing the lowest level of As was the organelle fraction. These results suggest that As accumulates in the pinna where it is primarily distributed in the cytoplasmic supernatant fraction. The role of As compartmentalization may be intricately linked with As detoxification in P. vittata L.