Abstract Cell survival is dependent upon precise control of the activity of the tumor suppressor p53. p53 is a transcription factor and is known to control a number of biological processes including cell cycle arrest and apoptosis by transactivating downstream genes. Several proteins are reported to affect p53 activity but the murine double-minute protein, MDM2, and a closely related family member, MDM4, are two of the most critical regulators of p53. In previous work, we have identified alternative splicing of both the MDM2 and MDM4 genes in response to UV damage implicating alternative splicing in the cell's normal surveillance mechanism. However, the role of the splice variants in the DNA damage pathway remains elusive. To determine the role the alternative splice forms play in the p53 damage response, overexpression of the UV-induced isoforms, MDM2-ALT1 and MDM4-ALT2, was performed. Both MDM2-ALT1 and MDM4-ALT2 are capable of negatively regulating the full-length MDM2 and MDM4 full-length proteins thereby increasing p53 levels. Interestingly, each splice variant activates a distinct and unique subset of p53 target genes. An additional type of cellular stress, hypoxia, induces alternative splicing of MDM2 as well, but generates a different splice form known as MDM2-ALT2. This splice form activates a third unique profile of p53 target genes. Hence, the splicing status of the MDM2 and MDM4 genes is capable of dictating unique p53 programs. We are also currently investigating p53-independent functions for the damage-induced splice variants to better understand their role in the damage response and tumorigenesis. This work elucidates regulated splicing as a novel mechanism by which cellular injury can control the activity of p53 within the cell. By producing specific alternatively spliced variants of both MDM2 and MDM4, yet another layer of p53 regulation is initiated in order to fine-tune the cell's response to damage. Because MDM2 and MDM4 alternative splice variants are characteristic of numerous cancer types, this work indicates that the status of MDM2 and MDM4 splicing could be a consideration in determining treatment options for cancers for which p53 activation is a therapeutic strategy. 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 LB-489. doi:1538-7445.AM2012-LB-489