This study evaluated, in vitro, the cytotoxicity of six root canal sealers after 12, 24 and 72 h of contact time, using an endothelial ECV-304 cell line. The MTT assay was used for analysis of cell viability. Twelve specimens of each sealer were prepared and randomly assigned to 6 groups according to the commercial brands (n=4/time). A control group was also formed, which was not subjected to the contact with sealers. To assess the effects of sealers on endothelial cells, the specimens were placed in culture plate wells and incubated at 37°C with 5% CO 2 and 100% humidity. MTT assays were performed in quadruplicate after 12, 24 and 72 h of contact of the sealer specimens with monolayers. Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc test at a significance level of 5%. Analysis of absorbance in the experimental groups showed that GuttaFlow presented the lowest cytotoxicity, with a mean absorbance of 0.048, followed by Pulp Canal Sealer (0.038), Sealer 26 (0.038), Endo Densell (0.036) and Pulp Fill (0.035). The control group had a mean absorbance of 0.098. Based on the results, Endofill and GuttaFlow were the most and the least cytotoxic sealers, respectively.