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Assessing the retention of cell therapies following implantation is vital and often achieved by labelling cells with 2′-[18F]-fluoro-2′-deoxy-D-glucose (18F-FDG). However, this approach is limited by local retention of cell-effluxed radiotracer. Here, in a preclinical model of critical limb ischemia, we assessed a novel method of cell tracking using 3′-deoxy-3′-L-[18F]-fluorothymidine (18F-FLT); a clinically available radiotracer which we hypothesise will result in minimal local radiotracer reuptake and allow a more accurate estimation of cell retention. Human endothelial cells (HUVECs) were incubated with 18F-FDG or 18F-FLT and cell characteristics were evaluated. Dynamic positron emission tomography (PET) images were acquired post-injection of free 18F-FDG/18F-FLT or 18F-FDG/18F-FLT-labelled HUVECs, following the surgical induction of mouse hind-limb ischemia. In vitro, radiotracer incorporation and efflux was similar with no effect on cell viability, function or proliferation under optimised conditions (5 MBq/mL, 60 min). Injection of free radiotracer demonstrated a faster clearance of 18F-FLT from the injection site vs. 18F-FDG (p ≤ 0.001), indicating local cellular uptake. Using 18F-FLT-labelling, estimation of HUVEC retention within the engraftment site 4 hr post-administration was 24.5 ± 3.2%. PET cell tracking using 18F-FLT labelling is an improved approach vs. 18F-FDG as it is not susceptible to local host cell reuptake, resulting in a more accurate estimation of cell retention. [ABSTRACT FROM AUTHOR]