Dietary selenium has been implicated in the prevention of cancer and other diseases, but its safety and efficacy is dependent on the supplemented form and its metabolites. In this study, X-ray absorption spectroscopy (XAS) and X-ray fluorescence microscopy (XFM) have been used to investigate the speciation and distribution of Se and Cu in vivo. In kidneys isolated from rats fed a diet containing 5 ppm Se as selenite for 3 weeks, Se levels increased 5-fold. XFM revealed a strong correlation between the distribution of Se and the distribution of Cu in the kidney, a phenomenon that has previously been observed in cell culture (Weekley et al., JBIC, J. Biol. Inorg. Chem., 2014, DOI: 10.1007/s00775-014-1113-x). However, X-ray absorption spectra suggest that most of the Se in the kidney is found as Se-Se species, rather than Cu-bound, and that most of the Cu is bound to S and N, presumably to amino acid residues in proteins. Furthermore, SOD1 expression did not change in response to the high Se diet. We cannot rule out the possibility of some Cu-Se bonding in the tissues, but our results suggest mechanisms other than the formation of Cu-Se species and SOD1 upregulation are responsible for the highly correlated distributions of Se and Cu in the kidneys of rats fed high selenite diets.