[(11)C]Flumazenil is widely used in positron emission tomography (PET) studies to measure GABA(A) receptors in vivo in humans. Although several different methods have been applied for the quantification of [(11)C]flumazenil binding, the reproducibility of these methods has not been previously examined. The reproducibility of a single bolus [(11)C]flumazenil measurements was studied by scanning eight healthy volunteers twice during the same day. Grey matter regions were analyzed using both regions-of-interest (ROI) and voxel-based analysis methods. Compartmental kinetic modelling using both arterial and reference region input function were applied to derive the total tissue distribution volume (V(T)) and the binding potential (BP) (BP(P) and BP(ND)) of [(11)C]flumazenil. To measure the reproducibility and reliability of each [(11)C]flumazenil binding parameter, absolute variability values (VAR) and intraclass correlation coefficients (ICC) were calculated. Tissue radioactivity concentration over time was best modelled with a 2-tissue compartmental model. V(T) showed with all methods good to excellent reproducibility and reliability with low VARs (mean of all brain regions) (5.57%-6.26%) and high ICCs (mean of all brain regions) (0.83-0.88) when using conventional ROI analysis. Also voxel-based analysis methods yielded excellent reproducibility (VAR 5.75% and ICC 0.81). In contrast, the BP estimates using pons as the reference tissue yielded higher VARs (8.08%-9.08%) and lower ICCs (0.35-0.80). In conclusion, the reproducibility of [(11)C]flumazenil measurements is considerably better with outcome measures based on arterial input function than those using pons as the reference tissue. The voxel-based analysis methods are proper alternative as the reliability is preserved and analysis automated.