The design and synthesis of potential antitumor antimetabolites 2'-deoxy-2'-hydroxylaminouridine (2'-DHAU) and -cytidine (2'-DHAC) are described. We found that 2'-DHAC in neutral solution generated 2'-aminoxy radicals at room temperature. 2'-DHAC inhibited the growth of L1210 and KB cells, with IC50 values of 1.58 and 1.99 microM, respectively, more potently than 2'-DHAU, with IC50 values of 34.5 and 27.3 microM, respectively. 2'-DHAC was effective against 9 human cell lines, with IC50 values of in the micromolar range. The in vivo antitumor activity of 2'-DHAC was also examined using the mouse leukemia P388 model, which gave a T/C value 167%. Phosphorylation of 2'-DHAC by uridine/cytidine kinase was essential for its cytotoxicity, as suggested by a competition experiment using several common nucleosides. Inhibition of DNA synthesis was the predominant mechanism of action of 2'-DHAC, although it has a ribo-configuration.