Nucleolin is a multifunctional abundant nucleolar protein whose transcriptional expression is trans-activated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein has defined roles in ribosomal RNA processing and transcriptional regulation of various genes including rRNA. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its sub-nuclear re-localization. Thus, nucleolin has been demonstrated to have both positive and negative effects on cell proliferation. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2 and inhibits Hdm2-mediated degradation of p53 albeit independent of the ARF (Alternate Reading Frame) tumor suppressor protein. Nucleolin also reduces Hdm2 protein levels by an unknown mechanism when over-expressed in cells to levels found in a variety of cancer cells. Here we have delineated that multiple domains of both nucleolin and Hdm2 are involved in distinct nucleolin-Hdm2 interactions in unstressed cells. Our data indicates that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2, as determined by Far-Western as well as GST-binding assays in vitro. While the N-terminus of nucleolin was found associated with the Hdm2 RING domain, the nucleolin RBDs robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2. The C-terminal GAR (Glycine-Arginine Rich) domain of nucleolin, shown previously to be essential for association with p53, did not bind Hdm2 yet was found to stimulate Hdm2-mediated p53-ubiquitination. Conversely, the nucleolin RBD that strongly interacted with Hdm2 was found to inhibit the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. There was no significant effect of over-expression of nucleolin or its various domains on Hdm2-autoubiquitination in cells. Similarly, the phosphorylation status of nucleolin did not significantly influence its binding to Hdm2. As might be expected from a key rRNA processing factor, our findings suggest that the activity of nucleolin towards Hdm2 can be modulated to yield either p53 up- or down-regulation. Our studies can aid in the future design of small molecule-based cancer therapeutics that can stimulate the wild-type p53 checkpoint in variety of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4532.