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Recent U.S. government action to lower the maximum concentration levels (MCL) of total arsenic (As) (10 ppb) in drinking water has raised serious concerns about the agricultural use of As-containing biosolids such as poultry litter (PL). In this study, solid-state chemical speciation, desorbability, and total levels of As in PL and long-term amended soils were investigated using novel synchrotron-based probing techniques (microfocused (micro) synchrotron X-ray fluorescence (SXRF) and micro-X-ray absorption near-edge structure (XANES) spectroscopies) coupled with chemical digestion and batch experiments. The total As levels in the PL were as high as approximately 50 mg kg(-1), and As(II/III and V) was always concentrated in abundant needle-shaped microscopic particles (approximately 20 microm x 850 microm) associated with Ca, Cu, and Fe and to a lesser extent with S, Cl, and Zn. Post-edge XANES features of litter particles are dissimilar to those of the organo-As(V) compound in poultry feed (i.e., roxarsone), suggesting possible degradation/transformation of roxarsone in the litter and/or in poultry digestive tracts. The extent of As desorption from the litter increased with increasing time and pH from 4.5 to 7, but at most 15% of the total As was released after 5 d at pH 7, indicating the presence of insoluble phases and/or strongly retained soluble compounds. No significant As accumulation (< 15 mg kg(-1)) was found in long-term PL-amended agricultural surface soils. This suggests that As in the PL may have undergone surface and subsurface transport processes. Our research results raise concerns about long-term PL amendment effects on As contamination in surrounding soil-water environments.