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Angiogenesis is an important factor for flap viability. It has been reported that ozonated oil contributed to improved neovascularization in an acute cutaneous wound healing model. This study was undertaken to evaluate the effect of ozonated olive oil on vascular endothelial growth factor (VEGF)–mediated neovascularization of skin flaps in rats.A skin flap model was established in 21 rats and evaluated within 3 groups. No treatment was given to the rats in group 1. Olive oil and ozonated olive oil were topically applied (twice daily) to the flap surface for 7 days in groups 2 and 3, respectively. Immunohistochemical staining was performed to analyze the expressions of VEGF and CD34.The mean numbers of VEGF- and CD34-positive staining microvascular structures were 8.86 (SD, 1.35) and 10.29 (SD, 1.80) in group 1, 15.00 (SD, 1.41) and 15.57 (SD, 1.72) in group 2, and 25.14 (SD, 2.41) and 25.00 (SD, 2.16) in group 3. The VEGF and CD34 expressions in group 3 were significantly higher than those in group 2 (P < .001). Their expressions in group 2 were significantly higher than those in group 1 (P < .001).Both ozonated olive oil and olive oil improved neovascularization when they were topically applied on skin flaps. The effect of ozone was more prominent.