THE fractionation of stable copper (Cu) isotopes during uptake into plant roots and translocation to shoots can provide information on Cu acquisition mechanisms. ISOTOPE fractionation (65Cu/63Cu) and intact tissue speciation techniques (X-ray absorption spectroscopy, XAS) were used to examine the uptake, translocation and speciation of Cu in strategy I (tomato–Solanum lycopersicum) and strategy II (oat–Avena sativa) plant species. Plants were grown in controlled solution cultures, under varied iron (Fe) conditions, to test whether the stimulation of Fe-acquiring mechanisms can affect Cu uptake in plants. ISOTOPICALLY light Cu was preferentially incorporated into tomatoes (Δ65Cuwhole plant-solution = c. −1‰), whereas oats showed minimal isotopic fractionation, with no effect of Fe supply in either species. The heavier isotope was preferentially translocated to shoots in tomato, whereas oat plants showed no significant fractionation during translocation. The majority of Cu in the roots and leaves of both species existed as sulfur-coordinated Cu(I) species resembling glutathione/cysteine-rich proteins. THE presence of isotopically light Cu in tomatoes is attributed to a reductive uptake mechanism, and the isotopic shifts within various tissues are attributed to redox cycling during translocation. The lack of isotopic discrimination in oat plants suggests that Cu uptake and translocation are not redox selective. ; Brooke M. Ryan, Jason K. Kirby, Fien Degryse, Hugh Harris, Mike J. McLaughlin and Kathleen Scheiderich