Fatty acid oxidation (FAO) is a major energy-providing process with important implications in cardiovascular, oncologic, neurologic, and metabolic diseases. A novel 4-thia oleate analog, 18-18 F-fluoro-4-thia-oleate ( 18 F-FTO), was evaluated in relationship to the previously developed palmitate analog 16-18 Ffluoro-4-thia-palmitate ( 18 F-FTP) as an FAO probe. Methods: 18 F-FTO was synthesized from a corresponding bromoester. Biodistribution and metabolite analysis studies were performed in rats. Preliminary small-animal PET studies were performed with 18 F-FTO and 18 F-FTP in rats. Results: A practical synthesis of 18 F-FTO was developed, providing a radiotracer of high radiochemical purity (.99%). In fasted rats, myocardial uptake of 18 F-FTO (0.70 6 0.30% dose kg [body mass]/g [tissue mass]) was similar to that of 18 F-FTP at 30 min after injection. At 2 h, myocardial uptake of 18 F-FTO was maintained, whereas 18 F-FTP uptake in the heart was 82% reduced. Similar to 18 F-FTP, 18 F-FTO uptake by the heart was approximately 80% reduced at 30 min by pretreatment of rats with the CPT-I inhibitor etomoxir. Folch-type extraction analyses showed 70-90% protein-bound fractions in the heart, liver, and skeletal muscle, consistent with efficient trafficking of 18 F-FTO to the mitochondrion with subsequent metabolism to protein-bound species. Preliminary small-animal PET studies showed rapid blood clearance and avid extraction of 18 F-FTO and of 18 F-FTP into the heart and liver. Images of 18 F-FTO accumulation in the rat myocardium were clearly superior to those of 18 F-FTP. Conclusion: 18 F-FTO is shown to be a promising metabolically trapped FAO probe that warrants further evaluation.