The ability of heart and skeletal muscle (SM) to switch between fat and carbohydrate oxidation is of high interest in the study of metabolic diseases and exercise physiology. Positron emission tomography (PET) imaging with the glucose analog 2-[¹⁸F]fluoro-2-deoxy-glucose (¹⁸F-FDG) provides a noninvasive means to quantitate glucose metabolic rates. However, evaluation of fatty acid oxidation (FAO) rates by PET has been limited by the lack of a suitable FAO probe. We have developed a metabolically trapped oleate analog, ( Z)-18-[¹⁸F]fluoro-4-thia-octadec-9-enoate (¹⁸F-FTO), and investigated the feasibility of using ¹⁸F-FTO and ¹⁸F-FDG to measure FAO and glucose uptake, respectively, in heart and SM of rats in vivo. To enhance the metabolic rates in SM, the vastus lateralis (VL) muscle was electrically stimulated in fasted rats for 30 min before and 30 min following radiotracer injection. The responses of radiotracer uptake patterns to pharmacological inhibition of FAO were assessed by pretreatment of the rats with the carnitine palmitoyl-transferase-1 (CPT-1) inhibitor sodium 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate (POCA). Small-animal PET images and biodistribution data with ¹⁸F-FTO and ¹⁸F-FDG demonstrated profound metabolic switching for energy provision in the myocardium from exogenous fatty acids to glucose in control and CPT-1-inhibited rats, respectively. Uptake of both radiotracers was low in unstimulated SM. In stimulated VL muscle, ¹⁸F-FTO and ¹⁸F-FDG uptakes were increased 4.4- and 28-fold, respectively, and CPT-1 inhibition only affected ¹⁸F-FTO uptake (66% decrease). ¹⁸F-FTO is a FAO-dependent PET probe that may allow assessment of energy substrate metabolic switching in conjunction with ¹⁸F-FDG and other metabolic probes.