Silva EJNL, Herrera DR, Almeida JFA, Ferraz CCR, GomesBPFA, Zaia AA. Evaluation of cytotoxicity and up‐regulation of gelatinases in fibroblast cells by three root repair materials. International Endodontic Journal, 45, 815–820, 2012. Aim  To investigate the effects of root repair materials on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in 3T3 fibroblasts. Methodology  Fibroblasts (3T3, 3 × 105 cells per well) were incubated with elutes of calcium hydroxide (Biodinâmica, Ibiporã, PR, Brazil), EndoBinder (Binderware, São Carlos, SP, Brazil) and mineral trioxide aggregate (MTA) (Angelus, Londrina, PR, Brazil) for 24 h. The cytotoxicity of all root repair materials was determined using the MTT assay. Supernatants of cell cultures incubated with materials were collected after 24 h to determine the levels of MMP‐2 and MMP‐9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and Tukey’s test. Results  Cells secreted MMP‐2 after 24 h with calcium hydroxide inducing significantly greater MMP‐2 expression in relation to the control and the other root repair materials (P < 0.05). The cytotoxicity results revealed that there was no significant difference in the cell viability of MTA, EndoBinder and the control group. However, there was a significantly reduced cell viability of 3T3 fibroblasts in association with calcium hydroxide (P < 0.05). Conclusions  Calcium hydroxide was associated with significantly less cell viability when compared with EndoBinder and MTA. All materials had gelatinolytic activity for MMP‐2 with calcium hydroxide being associated with the greatest activity.