Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) shows particular promise as an antitumour agent against colorectal cancer. It is known that KP1019 reacts with human serum proteins, whereby the major amount binds to albumin (present in large excess) and a smaller amount to transferrin. It has been hypothesised that transferrin-mediated uptake by transferrin receptor expressing tumour cells may in part explain the apparent tumour selectivity of this compound. Circular dichroism spectroscopy and electrospray ionisation mass spectrometry studies demonstrate that two equivalents of KP1019 bind specifically to human apotransferrin, while additional amounts of the ruthenium complex bind unspecifically. Uptake studies in the transferrin receptor-expressing human colon carcinoma cell line SW480 revealed a higher cellular accumulation of KP1019 in comparison to a KP1019-transferrin adduct (2∶1), while the uptake of a KP1019–Fe(III)-transferrin conjugate (1∶0.3∶1) significantly exceeded that of KP1019, suggesting that iron binding is necessary to obtain a protein conformation which favours recognition by the transferrin receptors on the cell surface. Our study showed that KP1019 is transported into the cell by both transferrin-independent and transferrin-dependent mechanisms. Transferrin-mediated uptake is more efficient when transferrin is saturated with iron to a physiological degree (∼30%). Cell fractionation experiments demonstrated that after a 2 h treatment of human colon cancer cells with 10 µM KP1019 on average 55% of the intracellular ruthenium is located in the cellular nucleus, while 45% remain in the cytosol and other cellular components.