[18F]AV-133 is a novel PET tracer for targeting the vesicular monoamine transporter 2 (VMAT2). The aim of this study is to characterize and quantify the loss of monoamine neurons with [18F]AV-133 in the MPTP-lesioned PD mouse model using animal PET imaging and ex vivo quantitative autoradiography (QARG). Methods: Optimal imaging time window of [18F]AV-133 was first determined in normal C57BL/6 mice (n = 3) with a 90-min dynamic scan. The reproducibility of [18F]AV-133 PET imaging was evaluated by performing a test–retest study within 1 week for the normal group (n = 6). For MPTP-lesioned studies, normal, and MPTP-treated [25 mg mg/kg once (Group A) and twice (Group B), respectively, daily for 5 days, i.p., groups of four normal and MPTP-treated] mice were used. PET imaging studies at baseline and at Day 4 post-MPTP injections were performed at the optimal time window after injection of 11.1 MBq [18F]AV-133. Specific uptake ratio (SUr) of [18F]AV-133 was calculated by [(target uptake–cerebellar uptake)/cerebellar uptake] with cerebellum as the reference region. Ex vitro QARG and immunohistochemistry (IHC) studies with tyrosine hydroxylase antibody were carried out to confirm the abundance of dopaminergic neurons. Results: The variability between [18F]AV-133 test–retest striatal SUr was 6.60 ± 3.61% with less than 5% standard deviation between animals (intervariability). The percentages of MPTP lesions were Group A 0.94 ± 0.29, −42.1% and Group B 0.65 ± 0.09, −60.4%. By QARG, specific binding of [18F]AV-133 was reduced relative to the control groups by 50.6% and 60.7% in striatum and by 30.6% and 46.4% in substantia nigra (Groups A and B, respectively). Relatively small [18F]AV-133 SUr decline was noted in the serotonin and norepinephrine-enriched regions (7.9% and 9.4% in mid-brain). Results obtained from IHC consistently confirmed the sensitivity and selectivity of dopaminergic neuron loss after MPTP treatment. Conclusions: [18F]AV-133 PET SUr displayed a high test–retest stability. The SUr significantly declined in the caudate putamen but not in the hypothalamus and midbrain regions after MPTP treatment in the mouse brain. The results obtained for QARG and IHC were consistent and correlated well with the PET imaging studies. On the basis of these concordant results, we find that [18F]AV-133 should serve as a useful and reliable PET tracer for evaluating nigrostriatal degeneration. Synapse 66:823–831, 2012. © 2012 Wiley Periodicals, Inc.