Molecular imaging of angiogenesis requires a highly specific and efficient contrast agent for targeting activated endothelium. We have previously demonstrated that paramagnetic and fluorescent liposomes functionalized with two angiogenesis-specific ligands, the galectin-1-specific anginex (Anx) and the a vß 3integrin-specific RGD, produce synergistic targeting effect in vitro. In the current study, we applied Anx and RGD dual-conjugated liposomes (Anx/RGD-L) for angiogenesis-specific MRI in vivo, focusing on the specificity and efficacy of liposome association with tumor endothelium. The targeting properties, clearance kinetics and biodistribution of Anx/RGD-L were investigated in B16F10 melanoma-bearing mice, and compared to liposomes functionalized with either Anx (Anx-L) or RGD (RGD-L). The contrast enhancement produced by dual- and single-targeted nanoparticles in the tumor was measured using in vivo T 1-weighted MRI, complemented by ex vivo immunohistochemical evaluation of tumor tissues. Blood clearance kinetics of Anx/RGD-L was three-fold more rapid than for RGD-L, but comparable to Anx-L. Both dual- and single-targeted liposomes produced similar changes in MRI contrast parameters in tumors with high inter-tumor variability (¿R 1 = 0.04 ± 0.03 s -1, 24 h post-contrast). Importantly, however, the specificity of Anx/RGD-L association with tumor endothelium of 53 ± 6%, assessed by fluorescence microscopy, was significantly higher compared to 43 ± 9% (P = 0.043) and 28 ± 8% (P = 0.0001) of Anx-L and RGD-L, respectively. In contrast, long-circulating RGD-L were on average 16% more efficient in targeting tumor endothelium compared to Anx/RGD-L. Significant differences were also found in the biodistribution of investigated contrast agents. In conclusion, synergistic targeting of a vß 3 and galectin-1 improved the specificity of the association of the liposomal contrast agent to tumor endothelium in vivo, providing therefore a more reliable MRI readout of the angiogenic activity. © 2011 Elsevier B.V. All rights reserved.