Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O₂) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O₂) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1^−/− murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1^+/+ MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase–specific cell killing. In support of this proposed mode of action, PARP inhibitor–treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated “contextual synthetic lethality.” As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations. Cancer Res; 70(20); 8045–54. ©2010 AACR.