Various metals are added to poultry diets to facilitate weight increase and disease prevention. The large amounts of poultry waste produced annually are dispersed intensively over relatively small areas of land, resulting in accumulations that pose potential environmental risks to the surface and groundwater. The focus of this study was to assess the distribution of heavy metals among various solid-phase fractions in soil profiles from a 25-year poultry waste-amended soil. Copper and Zn accumulated close to the soil surface where the total amounts of Cu and Zn in waste-amended soils were significantly higher than in nonamended soils. The total metal concentrations in amended soils were not critically high. Copper in the amended soil was present mostly in the organic matter (OM) fraction (46.9%), whereas Zn was found in the easily reducible oxide (ERO) fraction (47.3%). This suggests that the Cu and Zn in this long-term amended soil are potentially bioavailable and mobile. We observed the mobility of Zn through much of the soil profile of the long-term waste-amended soil. Zinc in this soil profile was found primarily in forms of the residual (RES) and crystalline iron oxide bound (CryFe) fractions, followed by the organic matter-bound and exchangeable (EXC) fractions.