Abstract Chimeric antigen receptor (CAR) T cell therapy has proven to be highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. These efforts face multiple challenges, including off-tumor target expression and checkpoint inhibition of CAR T cell activity in the tumor microenvironment. CAR T cells are much more potent compared to antibody therapeutics, therefore there is a need for more stringent CAR T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. In this study we employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model, which expresses both BCMA and ROR1. The anti-ROR1 CAR T cells used in this study recognized both the mouse and human proteins whereas the anti-BCMA CAR T cells recognized only the human protein. RNA ISH revealed that BCMA was only expressed in the xenograft tumor and in no mouse organs, while ROR1 was found to be expressed in the xenograft tumor as well as at low levels in mouse lung and liver. Duplex RNA ISH assay with probes targeting the CAR 3’ UTR and either IFNG or GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from vehicle, anti-ROR1 CAR T cell, or anti-BCMA CAR T cell treated mice. Activated anti-BCMA CAR T cells expressing GZMB and IFNG were found only in the xenograft tumor, where BCMA was expressed. In contrast, activated anti-ROR1 CAR T cells were found almost exclusively in mouse lung and liver with very few anti-ROR1 CAR T cells being found in the xenograft tumor, consistent with the previously observed pulmonary and hepatic toxicity of anti-ROR1 CAR T cells that was not predicted by IHC analysis of ROR1 protein presumably due to lack of antibody sensitivity. Lastly, we employed a multiplex ISH-IHC approach to confirm the presence of activated anti-BCMA CAR T cells in the xenograft tumor through simultaneous detection of the BCMA CAR 3’ UTR, IFNG, GZMB, and CD3, validating the anti-tumor activity of anti-BCMA CAR T cells. These data thus demonstrate how the RNAscope ISH assay can be utilized for CAR T efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR T cell pharmacodynamics and activation in tumor models. This technology has equal utility in understanding both CAR T and TCR-T cell activity in patient tumors. Citation Format: Helly Pimentel, Helen Jarnagin, Hailing Zong, Courtney Todorov, Kenneth Ganley, Fay Eng, Kevin Friedman, Molly Perkins, Shannon Grande, Courtney M. Anderson, Bingqing Zhang, Christopher Bunker, James B. Rottman, Xiao-Jun Ma. Preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2324.