Background: While representing only 3% of thyroid malignancies, medullary thyroid cancer (MTC) accounts for 14% of thyroid cancer deaths. MTC has a high rate of recurrence and lacks effective treatments. The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) activate the Notch1 signaling pathway, while lithium chloride inhibits the glycogen synthase kinase-3ß (GSK-3ß) pathway. These compounds have been shown to limit growth and suppress hormonal secretion; thus, targeting different signaling pathways may be an effective treatment. Methods: MTC cells were treated with varying combinations of up to 20 mM lithium chloride with either 3 mM VPA or 20 μM SBHA for 48 h. Western analysis was used to measure the effects on Notch1, GSK-3ß, and neuroendocrine (NE) markers. Growth was assessed by a methylthiazolyldiphenyl-tetrazolium (MTT) bromide cellular proliferation assay. Western analysis was used to determine the mechanism of growth regulation. Results: Combination therapy increased active Notch1, inhibited the GSK-3ß pathway, and decreased NE markers. Additive inhibition of growth was observed with combination therapy. Lower-dose combination therapy achieved greater decreases on NE markers and growth than treatment with any of the drugs alone. Moreover, an increase in the cleavage of the apoptotic markers caspase-3 and PARP was observed. Conclusions: Combination therapy with lithium chloride and HDAC inhibitors suppresses NE markers and decreases growth via apoptosis of MTC cells in vitro. With the possibility of increased efficacy and decreased toxicity, combination therapy may represent a new strategy to treat MTC. © 2010 Elsevier Inc. All rights reserved.