OBJECTIVE This study evaluated the cytotoxicity of the new experimental self-adhesive, methachrylate-based hybrid root canal sealer XT and compared it with the epoxy resin-based AH Plus Jet. METHODS The cytotoxicity of the tested materials was evaluated after 1, 24, 48, and 72 hours by using growing and confluent mouse fibroblast cell line L929. L929 fibroblasts were maintained in Dulbecco modified medium containing 10% fetal calf serum at 37°C and 5% CO2. At confluence, cells were seeded in 24-well plates at concentration of 1.5 × 10(5) cells (growing cells) or 2.5 × 10(5) (confluent cells) for each well. An amount of 5 μL of each root sealer (mixed according to manufacturer's specifications) was placed into individual wells containing a monolayer of L929 cells to mimic the in vivo condition of the possible extrusion of sealer in the periapical tissues. Neutral Red and [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] were used for the cytotoxicity evaluation. Untreated cells were used as control. Two-way analysis of variance with Bonferroni test was used to compare the toxicity of the 2 sealers; one-way analysis of variance with Tukey test was performed to compare the cytotoxicity of each sealer at any considered time points (P < .05). Results were confirmed by examination with optical microscope. RESULTS Both sealers induced a time-dependent cell death of mouse fibroblast L929; however, XT was less cytotoxic than AH Plus Jet as indicated by viability and morphologic analyses, and its initial cytotoxicity decreased progressively over time. CONCLUSIONS These data support the possible use of XT as an endodontic sealer.