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EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and functions in transcription regulation and RNA metabolism. Recent biochemical and EWSR1-depletion studies demonstrated that EWSR1 regulates critical phosphorylation events that control basal transcription. In Ewing sarcoma (EWS) cells, the interaction of EWSR1 and the fusion oncoprotein EWS-FLI1 results in EWSR1 no longer functioning as an effective regulator of transcription, which following DNA damage, enhances R-loop formation. To further elucidate EWSR1’s function in EWS and its contribution to EWS-FLI1’s deregulation of gene expression, we have generated reporter systems to visualize and quantify its endogenous expression within EWS cells. We used CRISPR-Cas9 and sgRNAs targeting sequences at the 5’ end of the first exon of EWSR1 to insert a fluorescent mNeonGreen (mNG) reporter gene into the EWSR1 loci of A673 or TC-32 EWS cell lines. We employed sequencing and RNAi-based analysis to identify and validate successfully modified clones. All modified clones harbored insertions into the unrearranged EWSR1 allele. We then employed super-resolution confocal microscopy to assess EWSR1’s localization in EWS cells. Analysis of modified EWS-cells showed mNG-EWSR1 forms puncta, restricted to the nucleoplasm, consistent with a nuclear protein in an active state. A subset of puncta exhibits a high density (HD) mNG-EWSR1 signal, defined by fluorescence at least twice the background signal. Our results show minimal colocalization of mNG-EWSR1 (total or HD) and a marker of chromatin accessibility, H3K27Ac. A small percentage (~5%) of total Ser5-phosphorylated RNA-pol II (pS5-RP-II), a marker of transcription initiation, colocalizes with total mNG-EWSR1, but critically the HD mNG-EWSR1 puncta all colocalize with pS5-RP-II. About 20% of total Ser2-phosphorylated RNA-pol II (pS2-RP-II), a marker of transcription elongation colocalizes with total mNG-EWSR1. As observed for pS5-RP-II, 100% of HD-mNG-EWSR1 puncta colocalize with pS2-RP-II. Finally, when we examined nuclear speckle structures (SC35/SRSF2 >200 µm), we observed a 20% overlap in their signals. We have seen comparable results using the mNG-EWSR1 expressing A673 cells. These findings demonstrate image-based quantification of endogenous EWSR1’s colocalization with RNA-polymerase II within EWS cells. Overall, EWSR1 colocalizes with about 20% of RNA-pol II in a state consistent with active transcription, and over 90% of high-density EWSR1 colocalizes with phosphorylated RNA pol II. Ongoing studies will assess changes in the distribution of EWSR1’s interactions with different proteins following inhibition of RNA-pol II phosphorylation, alterations in EWS-FLI1 expression, or the disruption of low complexity domain interactions. We anticipate findings from these studies will offer critical insights into the functional interactions that EWSR1 contributes to regulating gene expression in EWS cells. Citation Format: Natasha J. Caplen, Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Michael J. Kruhlak, Jan Wisniewski, Patricio Pichling, Katelyn R. Ludwig, Javed Khan, Raj Chari. Visualization of EWSR1’s colocalization with phosphorylated RNA-Polymerase II reveals its concentration at a subset of active regions of transcription in ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B012.