Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded beta-sheet fold. Previously, directed evolution of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin led to the identification of a mutant that bound to tumor-specific alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity. The objective of this study was to prepare and evaluate a radiofluorinated version of this knottin (termed 2.5D) for microPET imaging of integrin positive tumors in living subjects. Knottin peptide 2.5D was prepared by solid-phase synthesis and folded in vitro, and its free N-terminal amine was reacted with N-succinimidyl-4-18/19F-fluorobenzoate (18/19F-SFB) to produce the fluorinated peptide 18/19F-FB-2.5D. The binding affinities of unlabeled knottin peptide 2.5D and 19F-FB-2.5D to U87MG glioblastoma cells were measured by competition binding assay using 125I-labeled echistatin. It was found that unlabeled 2.5D and 19F-FB-2.5D competed with 125I-echistatin for binding to cell surface integrins with IC(50) values of 20.3 +/- 7.3 and 13.2 +/- 5.4 nM, respectively. Radiosynthesis of 18F-FB-2.5D resulted in a product with high specific activity (ca. 100 GBq/micromol). Next, biodistribution and positron emission tomography (PET) imaging studies were performed to evaluate the in vivo behavior of 18F-FB-2.5D. Approximately 3.7 MBq 18F-FB-2.5D was injected into U87MG tumor-bearing mice via the tail vein. Biodistribution studies demonstrated that 18F-FB-2.5D had moderate tumor uptake at 0.5 h post injection, and coinjection of a large excess of the unlabeled peptidomimetic c(RGDyK) as a blocking agent significantly reduced tumor uptake (1.90 +/- 1.15 vs 0.57 +/- 0.14%ID/g, 70% inhibition, P < 0.05). In vivo microPET imaging showed that 18F-FB-2.5D rapidly accumulated in the tumor and quickly cleared from the blood through the kidneys, allowing excellent tumor-to-normal tissue contrast to be obtained. Collectively, 18F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast and further demonstrates that knottins are excellent peptide scaffolds for development of PET probes with potential for clinical translation.