αVβ3 Integrins are a widely recognized target for in vivo molecular imaging of pathological conditions such as inflammation, cancer and rheumatoid arthritis. We have evaluated the sensitivity of a new, near-infrared fluorescence (NIRF), RGD cyclic probe (DA364) in noninvasive detection of αVβ3 integrin-overexpressing tumors. DA364's binding affinity for αVβ3 integrin was first evaluated in vitro. Human αVβ3 integrin-positive, U-87 MG glioblastoma cells were then xenografted in nude mice, and DA364 was injected intravenously (i.v.) to evaluate its in vivo distribution, specificity and sensitivity in comparison with a commercially available probe. DA364 bound αVβ3 integrin on U-87 MG cells with high affinity and specificity, both in vitro and in vivo. This binding specificity was corroborated by the strong inhibition of its tumor uptake induced by nonfluorescent, cyclic-RGD peptides. Ex vivo analysis showed that DA364 accumulated at the tumor site, whereas very low levels were detected in liver and spleen. In conclusion, DA364 allows sensitive and specific detection of transplantable glioblastoma by NIRF imaging, and is thus a promising candidate for the elaboration of imaging and therapeutic probes for αVβ3 integrin-overexpressing tumors. Copyright © 2011 John Wiley & Sons, Ltd.