The fern Pteris vittata accumulates unusually high levels of arsenic. Using X-ray absorption spectroscopy (XAS) and XAS imaging, we reveal the distribution of arsenic species in vivo. Arsenate is transported through the vascular tissue from the roots to the fronds (leaves), where it is reduced to arsenite and stored at high concentrations. Arsenic-thiolate species surrounding veins may be intermediates in this reduction. In gametophytes, arsenite is compartmentalized within the cell vacuole. Arsenic is excluded from cell walls, rhizoids, and reproductive areas. This study provides important insights into arsenic hyperaccumulation, which may prove useful for phytoremediating arsenic-contaminated sites, and demonstrates the strengths of XAS imaging for distinguishing highly localized species. ; Ingrid J. Pickering, Luke Gumaelius, Hugh H. Harris, Roger C. Prince, Gregory Hirsch, Jo Ann Banks, David E. Salt, and Graham N. George ; Copyright © 2006 American Chemical Society