To date there has been very little data on the cytotoxic response of different cell lines to root canal sealers. The objective of this study was to determine the cytocompatibility of three different extracts of root canal sealers and to compare the cytotoxic response of these materials on two different primary human oral fibroblasts (derived from gingiva and buccal mucosa) and one permanent hamster cell line (V79 cells). Cytotoxicity was judged using an assay of tetrazolium bromide reduction. The results showed that root canal sealers (AH plus, Canals, and N2) were cytotoxic to primary human oral fibroblast cultures and V79 cells. It was found that N2 was the most toxic root canal sealer among those tested in all cultures. The toxicity decreased in an order of N2 > AH plus approximately = Canals. The sensitivity of toxicity depended on the materials tested and the cell culture system used.