Objectives: Mitochondrial membrane potential (MMP) dependent enhanced uptake of phosphonium salts, including [³H]tetraphenylphosphonium (TPP), in myocardial cells, suggests the potential use of phosphonium salts as a tracer for cardiac imaging. In this study, we characterize the accumulation of TPP in myocardial cells and compare it with clinical counterpart, such as [¹⁸F]fluorodeoxyglucose (FDG) or [^99mTc]tetrofosmin (TF) in in vitro and in vivo. Methods: TPP accumulation was assessed in cell culture in different glucose concentrations with rat cardiomyoblast cell line; H9c2. To assess cellular uptake of tracers in different plasma (PMP) or mitochondrial membrane potential (MMP), PMP and MMP were altered using low/high K⁺buffer or some ionophores such as valinomycin and CCCP. Biodistribution studies and digital whole body autoradiography (DWBA) were performed using nude mice receiving 200-250 µCi FDG or TF and 5-10 µCi TPP intravenously. Results: Cellular uptake of TPP in H9c2 was about 30 times higher than that of TF. Reduction of tracer uptake in H9c2 cells after incubation in high K+ buffer was 70% for TPP and 30% for TF. Further reduction of tracer uptake after addition of valinomycin and CCCP was about 25%, 60% for TPP and 20 %, 22% for TF, respectively. Biodistribution of TPP in normal nude mice (n=12) showed relatively high accumulation in heart (8.3짹1.4%ID/g) and kidneys (17.8짹3.5%ID/g) but low uptake in other tissues (0.04-3.2%ID/g) at 1 hour of injection. TPP exhibited very low values in blood as early as 10 min, reflecting rapid washout from blood. DWBA images of mouse and rat showed low background activity and high radioactive signals from heart. Conclusion: The sensitive accumulation of TPP in terms of altered PMP and/or MMP in myocardial cells warrants further investigation of radiolabeled phosphonium analogs for imaging cardiac oxidative metabolism.