Abstract Aim/Introduction: [18F]fluorodeoxyglucose-positron emission tomography ([18F]FDG-PET) has been used as an imaging methods for measuring interscapular brown adipose tissue (iBAT) activity. However, [18F]FDG-PET has limitations for obtaining iBAT-specific images due to off-target effects with high uptake in skeletal muscle, tumors, and inflamed tissue. Uncoupling protein 1 (UCP1), a well-known biomarker for BAT, located in mitochondria also has been suggested as BAT imaging marker. Recently, UCP1 ThermoMouse was established as a reporter mouse for monitoring UCP1 expression. Translocator protein-18 kDa (TSPO) located in the mitochondria outer membrane along with UCP1 is overexpressed in BAT. TSPO targeting probe has been proposed as a potential imaging marker for iBAT imaging. To date, there have been no studies for BAT imaging using TSPO-targeting probe in UCP1 ThermoMouse reflecting UCP1 expression. Moreover, Cerenkov luminescence imaging (CLI) which captures Cerenkov radiation emitted from PET probes has been suggested as an alternative option for PET. In this study, we aim to evaluate [18F]fm-PBR28-d2 as a TSPO-targeting probe for iBAT imaging using PET and CLI in the UCP1 ThermoMouse. Materials and Methods: UCP1 ThermoMouse with insertion of a Luc2-T2A-tdTomato cassette into the initiation of codon of the Ucp1-coding sequence exon 1 were used to monitor UCP1 expression. UCP1 ThermoMouse were characterized with Western blotting and immunohistochemistry to measure the levels of UCP1 expression. PET images were acquired with SimPET, and optical images such as bioluminescence imaging (BLI), fluorescence imaging (FLI) and Cerenkov luminescence imaging (CLI) were acquired with IVIS 100. Results: BLI or FLI reflecting UCP1 expression were clearly showed iBAT in UCP1 ThermoMouse. Also, UCP1 and TSPO expression were correlated in iBAT. TSPO-targeting PET probe with [18F]fm-PBR28-d2 showed iBAT-specific signals without off-target effect in brain or heart. In addition, [18F]fm-PBR28-d2 better reflected individual variation in different UCP1 expression than [18F]FDG. In particular, higher molar activity of [18F]fm-PBR28-d2 was required to obtain better imaging quality of CLI as well as PET. In physiological changes such as cold stimulation and isoflurane exposure, we observed increased imaging signals with cold exposure and decreased imaging signals with prolonged isoflurane exposure. Conclusions: We showed that the TSPO-targeting probe is superior to [18F]FDG as a reliable imaging probe reflecting UCP1 expression in iBAT imaging. Probes with higher molar activity provide better images and are required for quantitative analysis of TSPO-CLI and PET. Our data suggest that CLI with TSPO-targeting probe could be an alternative option for PET. Citation Format: Seok-Yong Lee, Ho Rim Oh, Young-Hwa Kim, Sung-Hwan Bae, Yongseok Lee, Yun-Sang Lee, Byung Chul Lee, Gi Jeong Cheon, Keon Wook Kang, Hyewon Youn. Cerenkov luminescence imaging of interscapular brown adipose tissue using TSPO-targeting probe to overcome off-target effect of [18F]FDG. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5046.