Abstract The aim of this study was to evaluate in vitro the cytotoxicity on human periodontal ligament fibroblasts of three root-end filling materials: MTA Angelus(r), EndoSequence Root Repair Material Putty(r) and Super EBA(r). A primary culture of human periodontal ligament fibroblasts was previously obtained in order to evaluate the cytotoxicity of the three extracts from the root-end filling materials after 2 and 7 days of setting. Serial dilutions of these extracts (1:1, 1:2, 1:4 and 1:8) were evaluated at 1, 3 and 7 days using the methyl-thiazol-tetrazolium (MTT) colorimetric assay. Cell viability was evaluated as percentage of the negative control group, which represented 100% cell viability. Statistical analyses were done with t-test, ANOVA and Kruskal-Wallis test at a significance level of 5%. It was found that the main difference among root-end filling materials was in the higher dilutions (p<0.05), but there was a similar behavior in lower dilutions (p>0.05). Cell viability of MTA Angelus(r) was superior for 2-day setting (p<0.05), compared with the other two root-end fillings. There were no statistically significant differences between 7-day set MTA Angelus(r) and EndoSequence Root Repair Material Putty(r). Super EBA(r) showed the lowest percentage of cell viability at higher dilutions (p<0.05). Therefore, MTA Angelus(r) and EndoSequence Root Repair Material Putty(r) were less cytotoxic in the highest dilution (1:1) compared with Super EBA(r).