We have used copper-64-pyruvaldehyde-bis(N⁴-methylthiosemicarbazone) (⁶⁴Cu–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-[¹⁸F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. ⁶⁴Cu–PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to ⁶⁴Cu–PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with ⁶⁴Cu–PTSM than with FDG. However, both ⁶⁴Cu–PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The ⁶⁴Cu–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 ⁶⁴Cu–PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of ⁶⁴Cu–PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with ⁶⁴Cu–PTSM-labeled cells. Given the longer t_1/2 of ⁶⁴Cu (12.7 h) relative to ¹⁸F (110 min), longer cell-tracking periods (up to 24–36 h) should be possible now with PET.