The ruthenium (II) polypyridyl complexes (RPC), Δ-[(phen)₂Ru(tatpp)]Cl₂ (Δ-[3]Cl₂) and ΔΔ-[(phen)₂Ru(tatpp)Ru(phen)₂]Cl₄ (ΔΔ-[4]Cl₄, are a new generation of metal-based antitumor agents. These RPCs bind DNA via intercalation of the tatpp ligand, which itself is redox-active and is easily reduced at biologically relevant potentials. We have previously shown that RPC 4⁴⁺ cleaves DNA when reduced by glutathione to a radical species and that this DNA cleavage is potentiated under hypoxic conditions in vitro. Here, we show that 3²⁺ also exhibits free radical–mediated DNA cleavage in vitro and that 3²⁺ and 4⁴⁺ both exhibit selective cytotoxicity toward cultured malignant cell lines and marked inhibition of tumor growth in vivo. The murine acute toxicity of RPCs 3²⁺ and 4⁴⁺ (maximum tolerable doses ∼ 65 μmol/kg) is comparable with that for cisplatin (LD₅₀ ∼ 57 μmol/kg), but unlike cisplatin, RPCs are generally cleared from the body unchanged via renal excretion without appreciable metabolism or nephrotoxic side effects. RPCs 3²⁺ and 4⁴⁺ are shown to suppress growth of human non–small cell lung carcinoma (∼83%), show potentiated cytotoxicity in vitro under hypoxic conditions, and induce apoptosis through both intrinsic and extrinsic pathways. The novel hypoxia-enhanced DNA cleavage activity and biologic activity suggest a promising new anticancer pharmacophore based on metal complexes with aromatic ligands that are easily reduced at biologically accessible potentials. Mol Cancer Ther; 12(5); 643–53. ©2013 AACR.