Our objectives were 1) to characterize angiogenesis in the MDA-MB-435 xenograft mouse model with three-dimensional (3D) MR molecular imaging using α 5 β 1 (RGD)-or irrelevant RGS-targeted paramagnetic nanoparticles and 2) to use MR molecular imaging to assess the antiangiogenic effectiveness of α 5 β 1 (α ν β 3)-vs. α ν β 3-targeted fumagillin (50 μg/kg) nanoparticles. Tumor-bearing mice were imaged with MR before and after administration of either α 5 β 1 (RGD) or irrelevant RGS-paramagnetic nanoparticles. In experiment 2, mice received saline or α 5 β 1 (α ν β 3)-or α ν β 3-targeted fumagillin nanoparticles on days 7, 11, 15, and 19 posttumor implant. On day 22, MRI was performed using α 5 β 1 (α ν β 3)-targeted paramagnetic nanoparticles to monitor the antiangiogenic response. 3D reconstructions of α 5 β 1 (RGD)-signal enhancement revealed a sparse, asymmetrical pattern of angiogenesis along the tumor periphery, which occupied< 2.0% tumor surface area. α 5 β 1-targeted rhodamine nanoparticles colocalized with FITC-lectin corroborated the peripheral neovascular signal. α 5 β 1 (α ν β 3)-fumagillin nanoparticles decreased neovasculature to negligible levels relative to control; α ν β 3-targeted fumagillin nanoparticles were less effective (P> 0.05). Reduction of angiogenesis in MDA-MB-435 tumors from low to negligible levels did not decrease tumor volume. MR molecular imaging may be useful for characterizing tumors with sparse neovasculature that are unlikely to have a reduced growth response to targeted antiangiogenic therapy.—Schmieder, AH, Caruthers, SD, Zhang, H., Williams, TA, Robertson, JD, Wickline, SA, Lanza, GM Three-dimensional MR mapping of angiogenesis with α 5 β 1 (α ν β 3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model.