The PET radiotracer 18 F-(2S,4R)4-Fluoroglutamine ( 18 F-Gln) reflects glutamine transport and can be used to infer glutamine metabolism. Mouse xenograft studies have demonstrated that 18 F-Gln uptake correlates directly with glutamine pool size and is inversely related to glutamine metabolism through the glutaminase enzyme. To provide a framework for the analysis of 18 F-Gln-PET, we have examined 18 F-Gln uptake kinetics in mouse models of breast cancer at baseline and after inhibition of glutaminase. We describe results of the pre-clinical analysis and computer simulations with the goal of model validation and performance assessment in anticipation of human breast cancer patient studies. Methods: Triplenegative breast cancer and receptor-positive xenografts were implanted in athymic mice. PET mouse imaging was performed at baseline and after treatment with a glutaminase inhibitor or a vehicle solution for a total of four mouse groups. Dynamic PET images were obtained for one hour beginning at the time of intravenous injection of 18 F-FGln. Kinetic analysis and computer simulations were performed on a representative time-activity curves (TAC), testing 1-and 2-compartment models to describe kinetics.Results: Dynamic imaging for one hour captured blood and tumor TACs indicative of largely reversible uptake of 18 F-FGln in tumors. Consistent with this observation, a two-compartment model indicated a relatively low estimate of trapping (k 3 ) , suggesting that the one-compartment model is preferable. Logan plot graphical analysis demonstrated late linearity, supporting reversible kinetics and modeling with a single compartment. Analysis of the mouse data and simulations suggests that estimates of glutamine pools size, specifically the V D for 18 F-FGln, were more reliably estimated using the one-compartment reversible model compared to the two-compartment irreversible model. Tumor-to-blood ratios, a more practical potential proxy of V D , demonstrated good correlation with volume of distribution from singlecompartment models and Logan analyses. Conclusions: Kinetic analysis of dynamic 18 F-Gln-PET images demonstrated the ability to measure V D to estimate glutamine pool size, a key indicator of cellular glutamine metabolism, by both a one-compartment model and Logan analysis. Changes in V D with glutaminase inhibition supports the ability to assess response to glutamine metabolism-targeted therapy.Concordance of kinetic measures with tumor-to-blood ratios provides a clinically feasible approach for human imaging.