This study was designed to test the hypothesis that Roekoseal is less cytotoxic than eugenol-based Sealapex or calcium hydroxide-based Kerr's Pulp Canal Sealer. Embryonic, day-19, rat calvarial osteoblasts were cultured at 4 × 105 cells/well. The concentration of conditioned medium from each sealer producing 50% surviving cells (ED50) was determined and long-term viability and apoptosis of cells assessed after exposure to the ED50 of each sealer. Low concentrations of Kerr's Pulp Canal Sealer (190 mm2/1 ml) and high concentrations of Sealapex and Roekoseal (190 mm2/300 μl) produced the ED50. ED50 challenged osteoblasts had fewer viable cells at 72 h than at 24 h, with significantly more viable cells in the Roekoseal group. There was significantly less apoptotic activity in cells exposed to Roekoseal ED50 than in cells exposed to Kerr's Pulp Canal Sealer (p < 0.05) ED50. Roekoseal may be less cytotoxic than Kerr's Pulp Canal Sealer, but further studies of this new sealer are required.