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The aim of this study was to evaluate the cell viability and tissue reaction of NeoMTA Plus (NMP; Avalon Biomed Inc, Houston, TX) compared with mineral trioxide aggregate (MTA; Angelus, Londrina, PR, Brazil) and Biodentine (BD; Septodont, Saint-Maur-de-Fossés, France).Fibroblasts (3T3) were plated and exposed to 1% extract from the test material before and after setting. Cytotoxicity assessment was performed using the 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide and sulforhodamine B assays. In vivo evaluation consisted of polyethylene tube implantation of the materials in rat subcutaneous tissue. Histologic analysis occurred at 7, 30, and 90 days, scoring inflammatory events and collagen fiber formation. Analysis of variance and the Tukey and t tests were used for cytocompatibility assays, and the Kruskal-Wallis test followed by the Dunn test were used for biocompatibility assays (P ≤ .05).The materials in the cytotoxicity assays presented greater viability after setting (P ≤ .05). NMP and MTA presented higher viability than the control (Dulbecco modified Eagle medium) on the 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide assay before and after setting (P ≤ .05). The sulforhodamine B assay showed that MTA and BD presented less viability than NMP and the control, and NMP was similar to the control before setting. After setting, MTA and BD presented higher viability when compared with the control group (P ≤ .05), and NMP was similar to control. Inflammatory infiltrate reduction occurred throughout the test periods for all materials. At 7 days, neutrophils were present in BD (P ≤ .05), and granuloma and giant cells were present in BD and MTA. At 30 days, BD showed intense inflammatory infiltrates and a large number of macrophages when compared with NMP, MTA, and the control (P ≤ .05). At 90 days, BD presented a thick fiber layer compared with NMP (P ≤ .05).NMP showed similar biocompatible behavior to MTA and BD.NeoMTA Plus (NMP) had a suitable behavior on cell viability and in the tissue reaction parameters.Both in vitro and in vivo tests characterized the biocompatibility of NMP.NeoMTA Plus showed a biocompatible behavior similar to mineral trioxide aggregate and Biodentine.The inflammatory infiltrate decreased along the tested periods for all materials.The material extracts induced greater viability on fibroblast cells after setting.