This report compares tissue irritation by a newly developed calcium-based root canal filling material with irritation by previously available materials and evaluates the toxicity of various components in the root canal filling material. Polyethylene tubes filled with root canal filling materials were implanted in rat subcutaneous tissue, enucleated after 2, 4, 8 and 12 weeks, and histopathologically evaluated for tissue reaction. The tissue inflammation was microscopically evaluated and divided into 4 grades. The new root canal filling material has good tissue affinity compared to prior material. Transmission electronic microscopy and X-ray analysis revealed: titanium in AH-26; barium and bismuth in Canals, Tubli-seal and the newly developed root canal filling cement A; and zinc in the root canal filling cement B. Tissue was variously inflamed and, occasionally, necrosis was induced in the cells. The newly developed root canal filling cement A, showed better tissue affinity and no or slightly morphology change.