Abstract The tumor suppressor p53 can cause cell cycle arrest, DNA repair, and apoptosis via its function as a nuclear transcription factor. However, p53 also triggers apoptosis through a mitochondrial pathway. Mitochondrial p53 binds to anti-apoptotic (Bcl-2, Bcl-XL) and pro-apoptotic (Bak, Bax) proteins localized at the mitochondrial outer membrane (MOM), causing MOM permeabilization that eventually results in apoptosis. The purpose of this work was to develop an optimal version of p53 engineered with a mitochondrial targeting signal (MTS) to trigger rapid apoptosis via the intrinsic pathway. To further investigate mitochondrial activity of p53, we targeted p53 to specific mitochondrial compartments. EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane (“XL-MTS” from Bcl-XL; “TOM-MTS” from TOM20), inner membrane (“CCO-MTS” from cytochrome c oxidase) or matrix (“OTC-MTS” from ornithine transcarbamylase). The apoptotic mechanism for each construct was further studied using pifithrin-μ (which inhibits p53-mediated transcriptional activity), pifithrin-μ (which reduces binding of p53 to Bcl-2 and Bcl-XL), and by over-expressing the anti-apoptotic Bcl-XL. Apoptosis assays (caspase-9, TUNEL, Annexin-V, 7-AAD) were performed in T47D cells (human ductal breast epithelial tumor cell line). While all MTS-EGFP-p53 constructs showed apoptotic potential, only EGFP-p53-XL had the most significant apoptotic potential compared to its own EGFP-XL control. EGFP tagged to OTC, CCO and TOM tend to interfere with sensitive mitochondrial function versus EGFP-XL, which demonstrated minimal toxicity. Previous work in the literature found that targeting p53 to the mitochondrial matrix causes apoptosis. However, our work indicates targeting even non-toxic proteins (EGFP) to the matrix might cause some level of toxicity due to the MTS by itself. Further, we inquired if the apoptotic response was due to the Bcl-XL dependent pathway. CCO-EGFP-p53 initiated apoptosis mainly through the transcriptional activity of p53 (rescued by pifithrin-μ). TOM-EGFP-p53 is thought to cause apoptosis by activating the pro-apoptotic Bak (rescued by over-expressing Bcl-XL). OTC-EGFP-p53 resulted in apoptosis through binding to Bcl-XL (rescued by pifithrin-μ and over-expressing Bcl-XL) and showed additional apoptotic potential through transcriptional activity of p53 (rescued by pifithrin-μ). In addition, EGFP-p53-XL was the most specific to the p53/Bcl-XL mitochondrial pathway (rescued by pifithrin-μ and over-expressing Bcl-XL). Taken together our data highlights that targeting p53 to Bcl-XL via the XL MTS triggers a rapid apoptotic response through an interaction of p53 with anti-apoptotic Bcl-XL. Due to the ubiquitous role of p53, this gene therapy approach could be used in many different types of cancers. Acknowledgements: NIH CA151847 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1182. doi:1538-7445.AM2012-1182