Purpose: This study aimed at investigating binding specificity, suitability of reference regionbased kinetic modelling, and pharmacokinetics of the metabotropic glutamate receptor 1 (mGluR1) radioligand [ 11 C]ITDM in mice. Procedures: We performed in vivo blocking as well as displacement of [ 11 C]ITDM during positron emission tomography (PET) imaging using the specific mGluR1 antagonist YM-202074. Additionally, we assessed in vitro blocking of [ 3 H]ITDM at two different doses of YM-202074. As an alternative to reference region models, we validated the use of a noninvasive image-derived input function (IDIF) compared to an arterial input function measured with an invasive arteriovenous (AV) shunt using a population-based curve for radiometabolite correction and characterized the pharmacokinetic modelling of [ 11 C]ITDM in the mouse brain. Finally, we also assessed semi-quantitative approaches. Results: In vivo blocking with YM-202074 resulted in a decreased [ 11 C]ITDM binding, ranging from − 35.8 ± 8.0 % in pons to − 65.8 ± 3.0 % in thalamus. Displacement was also markedly observed in all tested regions. In addition, in vitro [ 3 H]ITDM binding could be blocked in a dosedependent manner. The volume of distribution (V T) based on the noninvasive IDIF (V T (IDIF)) showed excellent agreement with the V T values based on the metabolite-corrected plasma input function regardless of the metabolite correction (r 2 9 0.943, p G 0.0001). Two-tissue compartmental model (2TCM) was found to be the preferred model and showed optimal agreement with Logan plot (r 2 9 0.960, p G 0.0001). A minimum scan duration of 80 min was required for proper parameter estimation. SUV was not reliable (r 2 = 0.379, p = 0.0011), unlike the SUV ratio to the SUV of the input function, which showed to be a valid approach. Conclusions: No suitable reference region could be identified for [ 11 C]ITDM as strongly supported by in vivo and in vitro evidence of specific binding in all brain regions. However, by