Hypoxia-activated prodrugs (HAPs) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. The present study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacological properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 μM (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies whilst retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 to significantly correlate with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicates that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.