There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity (∼10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or ⁶⁴Cu-1,4,7,10-tetra-azacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC₅₀, ∼20 nmol/L) ⁶⁴Cu-DOTA–conjugated knottin peptides was 4.47% ± 1.21% and 4.56% ± 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC₅₀, ∼0.4 μmol/L; 1.48 ± 0.53%ID/g) and c(RGDyK) (IC₅₀, ∼1 μmol/L; 2.32 ± 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, ⁶⁴Cu-DOTA–conjugated knottin peptides generated lower levels of nonspecific liver uptake (∼2%ID/g) compared with c(RGDyK) (∼4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. ⁶⁴Cu-DOTA–conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers. [Cancer Res 2009;69(6):2435–42]