Copper (Cu) is an important environmental pollutant that exerts harmful effects on all living organisms when in excess. In an effort to remove thistoxin in situ, a bacterial Cu-binding protein gene CusF was engineered to target CusF for secretion to the cell wall and vacuolesand for accumulation in the cytoplasm. Analysis of transgenicArabidopsis plants showed that CusF was functionally active and that plants expressing cell wall- (CusF_cw transgenic lines) or vacuole-targeted CusF (CusF_vac transgenic lines) were more resistant to Cuexcess than untransformed plants and plants with cytoplasmic CusF(CusF_cyto transgenic lines). Under short-term (48 h) exposure to Cuexcess, CusF_cw transgenic lines showed up to 2-fold increased Cuaccumulation in roots compared with the untransformed plants; however, CusF_cyto lines and the wild-type plants had similar Cuconcentrations in both roots and shoots. Under long-term (40 d) exposure to Cu excess, all transgenic lines accumulated more Cu (up to3-fold) in roots than the untransformed plants, whereas only CusF_cytolines showed a marked increase (∼3-fold of the wild-type plants) of Cuaccumulation in shoots. In addition, expression of CusF in the cytosol dramatically enhanced Cu transport from roots to shoots when compared with plants with secretory pathway-targeted CusF. Our results demonstrate the feasibility of Cu tolerance and accumulation by engineering Cu-binding proteins targetable to subcellularcompartments and provide new insights into the multifaceted mechanisms of Cu partitioning between roots and shoots.