Objective:The aim of this in vitro study was to evaluate the cytotoxic effects of different glass ionomer cements applied to an immortalized odontoblast-cell line (MDPC-23). Methods:The cells were plated (30.000 cells/cm²) in wells of 24 well-dishes, and maintained in a humidified incubator for 72 hours at 37oC, 5% CO2 and 95% of air. Thirty round-shaped samples (2mm thick and 4mm in diameter) of each experimental and control materials were prepared: Group 1: Vitrebond (VTB, positive control); Group 2: Vitremer (VTM); Group 3: Rely-X luting cement (RX). In group 4, fresh culture medium (DMEM) was used. The samples were immersed in 1.1mL of DMEM and incubated for 24, 48 and 72 hours. Seventy-two hours after incubating the cells, the fresh DMEM was replaced by the conditioned DMEM, in which the samples were stored for the specific periods of time (24, 48, or 72 hours). Then the cells were incubated in contact to the conditioned DMEM for 24 hours. The cell metabolism was evaluated by the methyltetrazolium assay (MTT). Results:The numeric data were submitted to the statistical analysis of Kruskal-Wallis complemented by the Mann-Whitney test. It was demonstrated that VTB decreased the cell metabolism by 85% for all periods of time evaluated. On the other hand, at 24 hours exposure, VTM and RX decreased the mitochondrial respiration by 25,2% and 21,2%, respectively. At 48 hours exposure both experimental materials decreased the cell metabolism by 37,5% and 31,5%, respectively. No statistically significant difference was observed for both experimental materials at 48 and 72 hours exposure. Conclusions:It was concluded that VTM and RX present lower cytotoxic effects to the cultured odontoblast cell line when compared to VTB. The cytotoxicity of both experimental materials increased until 48 hours of exposure to the conditioned culture media.