OBJECTIVES: This study evaluated the biocompatibility in vivo and in vitro of MTA (Pro-Root®), Portland cement and modified Portland cement (gypsum added). METHOD: For the in vivo analysis, polyethylene tubes were implanted subcutaneously in rats. After 7, 14, 30 and 60 days the tissue specimens were prepared for histological examination. For cytotoxic analysis the materials were placed in contact with NIH-3T3 cells. After 1, 3, 5 and 7 days, the cells viability was analyzed. RESULTS: The histological analysis showed moderate inflammatory response at 7 days in all groups. After 14 days the control group, MTA and Portland cement showed a mild inflammatory process while modified Portland group showed moderate inflammatory process. After 30 and 60 days all materials showed scarce inflammatory infiltrate and fibrosis. All the substances permitted the cell growth throughout the 7 days of experiment and presented similar cell viability. CONCLUSIONS: According to these experimental conditions, all the tested materials were biocompatible.