Hypoxic cells of solid tumors are an obstacle to effective cancer therapy. Since hypoxic cells remote from the tumor blood supply may have a greater capacity for reductive reactions than well-oxygenated cells, we have prepared a series of anthraquinone prodrugs which may be capable of generating a reactive quinonemethide species following enzymatic reduction to the hydroquinone and loss of the substituent on the methylene group in the 2 position. The synthesized 2-methyl-substituted anthraquinone derivatives have first half-wave reduction potentials of -0.52 to -0.56 V at pH 7.0, which are the lowest oxidation-reduction potentials of quinone bioreductive alkylating agents synthesized by this laboratory to date. Tests of the cytotoxicity of these agents to oxygenated and chronically hypoxic EMT6 tumor cells in culture demonstrated that 2-(hydroxymethyl)anthraquinone, 2-[(N-methylcarbamyl)-methyl]anthraquinone, 2-[[(p-toluenesulfonyl)oxy]methyl]anthraquinone, and 2-(methoxymethyl)anthraquinone were significantly more toxic to hypoxic cells than to their normally aerated counterparts. The findings demonstrate differences between various leaving groups in the 2 position for the expression of differential cytotoxicity.