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The purpose of this study was to evaluate the cytocompatibility of two different types of root canal sealers in cell culture. Human periodontal ligament cells were cultured with set materials from an experimental glass-ionomer cement sealer (KT-308) and a commercially available zinc oxide–eugenol-based sealer (Canals) for 1, 3, and 7 days. Cytotoxic effects were evaluated from the morphological changes under a light microscope. Canals induced severe degenerative alteration of human periodontal ligament cells. In contrast, human periodontal ligament cells adjacent to KT-308 showed normal morphology and growth during the culture period. These results suggest that the glass-ionomer cement sealer, KT-308, is cytocompatible and has good potential as a root canal sealer.