Abstract Gap junctions are membrane channels allowing intercellular communication between adjacent cells, and regulate cellular processes such as metabolism, proliferation, and differentiation. Connexins (Cx), the constituent proteins of gap junctions, are aberrantly localized or have decreased expression in many cancers, and some connexins act as tumor suppressors when overexpressed. Connexins are also highly regulated during development and when overexpressed, some have been shown to enhance cellular differentiation. In the present study, the role of neuronal connexins, Cx36 and Cx45, in the differentiation of SH-SY5Y human neuroblastoma cells was investigated. Retinoic acid was used as the differentiating agent, as it is often employed in differentiation therapy of tumor cells. Retinoic acid induces neuritogenesis and expression of differentiation markers, such as growth associated protein 43 (Gap43), neuropeptide Y (NPY), and neuron specific enolase (NSE) in SH-SY5Y cells. Following 2, 4, and 10 days of retinoic acid induced differentiation of SH-SY5Y cells, Cx36 mRNA expression showed a significant increase compared to non-treated cells, while Cx45 mRNA expression remained stable. Cx36 was overexpressed using liposome-mediated transfection, and an increase in the mRNA levels of Gap43, NSE, and NPY was observed after 48 hours compared to control EGFP transfected cells. Cx36 overexpression also enhanced retinoic acid induced differentiation, as evidenced by the increase in mRNA expression of differentiation markers, Gap43 and NPY, following 2 days of treatment, and will now be confirmed following 4 and 10 days of treatment. Therefore, Cx36 overexpression induces and enhances the differentiation of SH-SY5Y neuroblastoma cells. Current studies involve knocking down Cx36 and Cx45 with shRNA and inducing differentiation of SH-SY5Y cells to determine whether connexin knock down diminishes neuritogenesis or differentiation marker expression. These studies provide insight into the role of connexins in the differentiation therapy approach to cancer treatment. 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 LB-386.