Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). Hydrogen peroxide (H₂O₂) has been shown to increase VEGF expression and to be involved in angiogenesis. We tested the hypothesis that pretreatment with H₂O₂ enhances the efficacy of BMCs for neovascularization. H₂O₂ pretreatment was done by incubating mouse BMCs in 5 µM H₂O₂ for 30 min, followed by washing twice with PBS. The H₂O₂-pretreated and untreated BMCs were then studied in vitro and in vivo. RT-PCR analysis showed that expression of VEGF and Flk-1 mRNA was significantly higher in H₂O₂-pretreated BMCs than in untreated BMCs, after 12 and 24 h culture (P < 0.01). Pretreatment with H₂O₂ also effectively enhanced the endothelial differentiation from BMCs, as shown by the percentage of VE-cadherin positive cells after 1 and 7 days of culture (P < 0.05). To estimate the angiogenic potency in vivo,H₂O₂-pretreated or untreated BMCs were intramuscularly implanted into the ischemic hindlimbs of mice. After 14 days of treatment, many of the H₂O₂-pretreated BMCs were viable, showed endothelial differentiation, and were incorporated in microvessels. Conversely, the survival and incorporation of the untreated BMCs was relatively poor. Microvessel density and blood flow in the ischemic hindlimbs were significantly greater in the mice implanted with H₂O₂-pretreated BMCs than in those implanted with untreated BMCs (P < 0.05). These results show that the short-term pretreatment of BMCs with low-dose H₂O₂ is a novel, simple, and feasible method of enhancing their angiogenic potency.