Abstract Purpose GluN2B containing N-methyl-D-aspartate receptors (NMDARs) play an essential role in neurotransmission and are a potential treatment target for multiple neurological and neurodegenerative diseases, including stroke, Alzheimer’s disease, and Parkinson’s disease. (R)-[¹⁸F]OF-Me-NB1 was reported to be more specific and selective than (S)-[¹⁸F]OF-Me-NB1 for the GluN2B subunits of the NMDAR based on their binding affinity to GluN2B and sigma-1 receptors. Here we report a comprehensive evaluation of (R)-[¹⁸F]OF-Me-NB1 and (S)-[¹⁸F]OF–Me-NB1 in nonhuman primates. Methods The radiosynthesis of (R)-[¹⁸F]OF-Me-NB1 and (S)-[¹⁸F]OF-Me-NB1 started from ¹⁸F-fluorination of the boronic ester precursor, followed by removal of the acetyl protecting group. PET scans in two rhesus monkeys were conducted on the Focus 220 scanner. Blocking studies were performed after treatment of the animals with the GluN2B antagonist Co101,244 or the sigma-1 receptor antagonist FTC-146. One-tissue compartment (1TC) model and multilinear analysis-1 (MA1) method with arterial input function were used to obtain the regional volume of distribution (V_T, mL/cm³). Occupancy values by the two blockers were obtained by the Lassen plot. Regional non-displaceable binding potential (BP_ND) was calculated from the corresponding baseline V_T and the V_ND derived from the occupancy plot of the Co101,244 blocking scans. Results (R)- and (S)-[¹⁸F]OF-Me-NB1 were produced in > 99% radiochemical and enantiomeric purity, with molar activity of 224.22 ± 161.69 MBq/nmol at the end of synthesis (n = 10). Metabolism was moderate, with ~ 30% parent compound remaining for (R)-[¹⁸F]OF-Me-NB1 and 20% for (S)-[¹⁸F]OF-Me-NB1 at 30 min postinjection. Plasma free fraction was 1–2%. In brain regions, both (R)- and (S)-[¹⁸F]OF-Me-NB1 displayed fast uptake with slower clearance for the (R)- than (S)-enantiomer. For (R)-[¹⁸F]OF-Me-NB1, both the 1TC model and MA1 method gave reliable estimates of regional V_T values, with MA1 V_T (mL/cm³) values ranging from 8.9 in the cerebellum to 12.8 in the cingulate cortex. Blocking with 0.25 mg/kg of Co101,244 greatly reduced the uptake of (R)-[¹⁸F]OF-Me-NB1 across all brain regions, resulting in occupancy of 77% and V_ND of 6.36, while 0.027 mg/kg of FTC-146 reduced specific binding by 30%. Regional BP_ND, as a measure of specific binding signals, ranged from 0.40 in the cerebellum to 1.01 in the cingulate cortex. Conclusions In rhesus monkeys, (R)-[¹⁸F]OF-Me-NB1 exhibited fast kinetics and heterogeneous uptake across brain regions, while the (S)-enantiomer displayed a narrower dynamic range of uptake across regions. A Blocking study with a GluN2B antagonist indicated binding specificity. The value of BP_ND was > 0.5 in most brain regions, suggesting good in vivo specific binding signals. Taken together, results from the current study demonstrated the potential of (R)-[¹⁸F]OF-Me-NB1 as a useful radiotracer for imaging the GluN2B receptors.