Abstract The cytotoxic effects on cultured rat bone cells of newly-developed root canal sealers and commercially available sealers were compared. Various root canal sealers were applied to cultured bone cells obtained from rat calvaria by the enzyme digestion method. Measurement of [3H]-thymidine incorporation, alkaline phosphatase activity, and calcium release were performed after 24 and 48 h. No significant difference was found in cellular DNA synthesis and alkaline phosphatase activity between cells exposed to New B-l, New B-5 and controls after exposure for 24 and 48 h. Cells in contact with Tubliseal, Diaket and AH-26 demonstrated a significant difference from controls in DNA synthesis and alkaline phosphatase activity. Calcium release at 24 h was significantly different in the cells treated with New B-1 and New B-5 than in controls. No appreciable difference was found, however, between New B-6, Sealapex and controls. At 48 h, cells treated with New B-l, New B-5 and New B-6 showed differed significantly difference from controls, but the cells exposed to Sealapex did not. The newly-developed root canal sealers had lower toxicity in vitro than five types of commercially available root canal sealers.