Detecting changes in receptor binding at the metabotropic glutamate receptor 5 (mGluR5) with the PET allosteric antagonist, [11C]ABP688, may be valuable for studying dysfunctional glutamate transmission associated with psychiatric illnesses. This study was designed to validate the findings of a recent pilot study in baboons which reported a significant global decrease from baseline [11C]ABP688 binding after increasing endogenous glutamate with 50 mg/kg N-acetylcysteine (NAC), with no change from test to retest. In rhesus monkeys (n = 5), paired [11C]ABP688 scans were performed on the same day on the Focus-220 as follows (n = 3 per group): test-retest, baseline-NAC (50 mg/kg), and baseline-NAC (100 mg/kg). Multiple modeling methods were evaluated for kinetic analysis to estimate the total volume of distribution (VT) and non-displaceable binding potential (BPND) in regions-of-interest (ROIs), with the cerebellum gray matter (CGM) as the reference region. There was an increasing trend from test to retest BPND across ROIs (13%). NAC (50 mg/kg and 100 mg/kg) increased VT (5% and 19%) and decreased BPND (3% and 10%), respectively, significant only for VT in ROIs at the 100 mg/kg dose. High intersubject variability in BPND was comparable to that reported in the baboon study. However, interpretability of BPND is difficult with increases in VT in the CGM reference region at the higher NAC dose. Additionally, the net reduction in BPND from the baseline-NAC scans may be obscured due to observed increases in test-retest BPND. Thus, we did not strictly replicate the findings in the baboon study based on BPND. Synapse 67:489–501, 2013. © 2013 Wiley Periodicals, Inc.