Abstract Introduction: There are four mammalian Notch receptors (Notch1-4), which exhibit unique and overlapping expression pattern and act as oncogenes or tumor suppressor genes depending on the cell and context type. We previously reported that Notch1 pathway is minimally active in neuroendocrine (NE) tumors. Furthermore, induction of the Notch1 signaling pathway by over- expression or by pharmacological treatment with valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) suppresses hormone secretion. However, the role of other Notch isoforms is not clear in carcinoid. In this study, we analyzed the presence of Notch isoforms and their regulatory role on chromogranin A (CgA), a clinically important NE marker, and achaete-scute complex-like1 (ASCL1), a known downstream target of Notch. Methods: Expression of four Notch isoforms was quantified by real time RT-PCR from parental BON cells (GI carcinoid) and VPA treated BON cells. pcDNA3.3 TOPO plasmid containing constitutively expressed human active portion of Notch3, NICD3, was transiently transfected into BON cells and the cell lysates were tested for the presence of NOTCH3. The levels of NE markers such as CgA and ASCL1 were analyzed by Western blot. Results: Gene expression analysis showed that the parental BON cells had very minimal levels of Notch1-3. Notch4 expression was not detected. In VPA treated cells, the levels of Notch1, 2 and 3 were increased significantly. Overall, the Notch3 expression level was higher than Notch1 and 2 in VPA treated cells. Importantly, there was a 60-fold induction of Notch3 expression in VPA treated cells compared to parental untreated cells. VPA treatment also resulted in a 15-fold and 20-fold increase for Notch1 and Notch2 respectively whereas Notch4 was not detected in these cells. The constitutive expression of NICD3 in BON cells resulted in a reduction in ASCL1 and CgA. Conclusions: Carcinoid tumor cells have a paucity of all Notch isoforms, including Notch3. For the first time, we demonstrate that Notch3 regulates the NE phenotype, and may play an important role in regulating carcinoid tumor progression. These findings warrant further investigation on targeting Notch3 for carcinoid cancer therapy. 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 3085.