Tenascin-C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that tenascin-C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting tenascin-C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral tenascin-C expression in vivo. To address the need for an agent to image and quantify tenascin-C, we report the development of a radioactive PET tracer based on a tenascin-C-specific single-stranded DNA aptamer (tenascin-C aptamer). Tenascin-C aptamer was radiolabeled with (18)F and (64)Cu. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of tenascin-C aptamer were performed in comparison to a nonspecific scrambled aptamer (Sc aptamer). The labeled tenascin-C aptamer provided clear visualization of tenascin-C-positive but not tenascin-C-negative tumors. The uptake of tenascin-C aptamer was significantly higher than that of Sc aptamer in tenascin-C-positive tumors. The labeled tenascin-C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. Our data suggest that suitably labeled tenascin-C aptamer can be used as a PET tracer to image tumor expression of tenascin-C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring.