We have used copper-64-pyruvaldehyde-bis( N 4 -methylthiosemicarbazone) ( 64 Cu–PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[ 18 F]-Fluoro-2-deoxy- d -glucose (FDG) cell labeling also was evaluated for comparison. 64 Cu–PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64 Cu–PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64 Cu–PTSM than with FDG. However, both 64 Cu–PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64 Cu–PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64 Cu–PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64 Cu–PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64 Cu–PTSM-labeled cells. Given the longer t 1/2 of 64 Cu (12.7 h) relative to 18 F (110 min), longer cell-tracking periods (up to 24–36 h) should be possible now with PET.