Copper imbalance is implicated in many diseases, including cancer. Copper in blood is mainly transported by carrier proteins but a small fraction is bound to low molecular weight species, possibly amino acids. Their roles in cellular copper delivery are unknown. Our aim was to test whether accumulation of 64Cu into cancer-derived cells can be influenced by copper-binding serum amino acids. In vitro cellular accumulation of 64Cu was measured in Hank's Balanced Salt Solution in the presence of 100 μM l-histidine, l-methionine, l-cysteine and l-threonine. l-Cysteine markedly increased 64Cu accumulation and retention in DU145, PC3 and SK-OV-3 cells, while some other cell lines did not show an effect. This effect was not due to 64Cu delivery in the form of a 64Cu-cysteine complex, nor to reduction of 64Cu(ii) to 64Cu(i) by l-cysteine. Pre-incubation of cells with l-cysteine increased 64Cu accumulation, even if l-cysteine was removed from HBSS before 64Cu was added. The effect of l-cysteine on 64Cu accumulation was not mediated by increased glutathione synthesis. Despite the demonstrable in vitro effect, pre-injection of l-cysteine precursor N-acetyl-cysteine (NAC) in vivo did not enhance 64Cu delivery to DU145 xenografts in mice. Instead, it decreased 64Cu accumulation in the DU145 tumour and in brain, as assessed by PET imaging. We conclude that 64Cu is not delivered to DU145 cancer cells in vitro as a complex with amino acids but its cellular accumulation is enhanced by l-cysteine or NAC influx to cells. The latter effect was not demonstrable in vivo in the DU145 xenograft.