Small molecule ruthenium complexes show great promise as anticancer pharmaceuticals, but further rational development of these as drugs is stymied by an incomplete understanding of the mechanisms that give rise to markedly different biological behaviour for structurally similar species. X-ray fluorescence imaging at two incident energies was used to reveal the intracellular distribution of Ru in single human cells treated with KP1019, showing Ru localised in both cytosol and in the nuclear region. In addition the imaging showed that treatment with KP1019 modulated Fe distribution to resemble the Ru distribution, without affecting cellular Fe content. In stark contrast, Ru could not be visualised in cells treated with NAMI-A, indicating that it was not internalised and supporting the proposition that its activity is exerted through a membrane-binding mechanism.