Alteration in mitochondrial transmembrane potential (ΔΨ(m)) is an important characteristic of cancer. The observation that the enhanced negative mitochondrial potential is prevalent in tumor cell phenotype provides a conceptual basis for development of mitochondrion-targeting therapeutic drugs and molecular imaging probes. Since plasma and mitochondrial potentials are negative, many delocalized organic cations, such as rhodamine-123 and (3)H-tetraphenylphosphonium, are electrophoretically driven through these membranes, and able to localize in the energized mitochondria of tumor cells. Cationic radiotracers, such as (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin, have been clinically used for diagnosis of cancer by single photon emission computed tomography (SPECT) and noninvasive monitoring of the multidrug resistance (MDR) transport function in tumors of different origin. However, their diagnostic and prognostic values are often limited due to their insufficient tumor localization (low radiotracer tumor uptake) and high radioactivity accumulation in the chest and abdominal regions (low tumor selectivity). In contrast, the (64)Cu-labeled phosphonium cations represent a new class of PET (positron emission tomography) radiotracers with good tumor uptake and high tumor selectivity. This review article will focus on our recent experiences in evaluation of (64)Cu-labeled phosphonium cations as potential PET radiotracers. The main objective is to illustrate the impact of radiometal chelate on physical, chemical, and biological properties of (64)Cu radiotracers. It will also discuss some important issues related to their tumor selectivity and possible tumor localization mechanism.