Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64 Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ( 64 Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124 I-2¢-fluoro-2¢-deoxy-1-b-d-arabinofuranosyl-5-iodouracil ( 124 I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with 64 Cu-PTSM or 124 I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptasepolymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124 I-FIAU (95.2% -1.1%) was fourfold higher than 64 Cu-PTSM (23.6% -0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124 I-FIAU and 64 Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64 Cu-PTSM-and 124 I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124 I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64 Cu-PTSM-labeled cell-tracking method is more efficient than 124 I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of 124 I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of 124 I-FIAU in vitro, the in vivo cell-tracking method using 64 Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.