Molecular imaging of vesicular acetylcholine transporter (VAChT) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT‐specific positron emission tomography radiotracer, ((−)‐(1‐(‐8‐(2‐fluoroethoxy)‐3‐hydroxy‐1,2,3,4‐tetrahydronaphthalen‐2‐yl)piperidin‐4‐yl)(4‐fluorophenyl)‐methanone) (18FVAT) in macaques. Total volume distribution (VT) estimates were compared for one‐tissue compartment model (1TCM), two‐tissue compartment model (2TCM), Logan graphic analysis (LoganAIF) and multiple linear analysis (MA1) with arterial blood input function using data from three macaques. Using the cerebellum‐hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM), simplified RTM (SRTM), and Logan graphic analysis (LoganREF). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for 18FVAT; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r > 0.93, p < 0.05). Test–retest studies demonstrated that 18FVAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate 18FVAT is a promising VAChT positron emission tomography tracer for quantitative assessment of VAChT levels in the brain of living subjects. PET imaging of vesicular acetylcholine transporter (VAChT) in brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease (AD). In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT‐specific PET radiotracer 18FVAT in macaques. The results demonstrated that 18FVAT is a promising VAChT PET tracer for quantitative assessment of VAChT levels in the brain of living subjects.