Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin αvβ3, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of 18F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with 18F in a simple one-pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with 18F at a specific activity of 1.8 MBq nmol−1 and a radiochemical purity of 100% could be achieved. The logP value of 18F-labeled NODAGA-E-[c(RGDfK)]2 was −4.26 ± 0.02. In biodistribution studies, 18F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g−1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of 18F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g−1, 2 h p.i.) was significantly lower than that of reference compounds 68Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g−1; p <0.001) and 111In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g−1; p < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with 18F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g−1). The αvβ3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with 18F-labeled NODAGA-E-[c(RGDfK)]2. In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with 18F using a direct aqueous, one-pot method and it accumulated specifically in αvβ3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.