The lipophilic cationic radiotracer (99m)Tc-sestamibi, known to be concentrated within mitochondria, is widely used for myocardial perfusion and to a lesser extent for muscle metabolism imaging. However, the exact distribution pattern in skeletal muscle has not been yet studied in detail. The present study aims to investigate the (99m)Tc-sestamibi uptake in rat skeletal muscle and myocardium in relation to their metabolic characteristics. (99m)Tc-sestamibi was i.v. administered in twenty adult male Wistar rats and uptake, as percent of injected dose per tissue gram (%ID/g), in the myocardium, soleus, extensor digitorum longus and gastrocnemius muscles was assessed 2 h after the injection. Muscle uptake was also correlated with myocardial uptake, muscle weight and body weight. Skeletal muscle (99m)Tc-sestamibi uptake was a small (9-16 %) fraction of that found in myocardium (1.71+/-0.63 %ID/g). Among the three hindlimb muscles considered, the slow-oxidative soleus muscle showed the highest uptake (0.28+/-0.16 %ID/g). Metabolically diverse parts of the gastrocnemius muscle showed different uptake. Skeletal muscle uptake was positively correlated with myocardial uptake and both were negatively correlated with tissue and body weight. Skeletal muscle and myocardium (99m)Tc-sestamibi uptake is related to their metabolic profile. Myocardium, with an exceptional rich mitochondrial concentration, shows much higher (99m)Tc-sestamibi uptake compared to skeletal muscles. Among muscles, uptake is dependent on their mitochondrial content. Evidence of matching exists between myocardial and muscle uptake, and both are size-dependent.