– The aim of this study was to evaluate the cytotoxic effect of four root canal sealers: AH26, AH Plus, Diaket and Apexit. In the experiment two cell lines, human cervical carcinoma (HeLa) cells and mouse skin fibroblasts (L929), were used. Under aseptic conditions, the sealers were prepared according to the manufacturers' directions, and 0.01 mL of each material was placed in a 24-well plate. The sealers were covered with cell suspension. The cytotoxicity was estimated by determining the number of viable cells by a light microscope, as well as the total number of cells 24 h, 48 h and 120 h after the treatment with mentioned materials. The results obtained in this study showed the high cytotoxcity of the new AH Plus root canal sealer, which was shown to be equally or more toxic to the standard AH26 and Diaket materials. Apexit was the least toxic sealer.