Objectives: Evaluate the bone formation in periodontal fenestration defects in rats applying bone marrow mesenchymal stem cells (MSC) with or without Enamel Matrix Derivate (EMD). Methods: Periodontal fenestration defects of 2 mm in height, 4 mm in width and 1 mm in depth were created in the mandible of isogenic rats. The animals were allocated into 6 groups and further divided into 3 subgroups for euthanasia at 15, 30 or 60 days postoperative (n=6 for each subgroup). Control Group (C) was the spontaneous healing. In the Propylene Glycol Alginate Group (PGA) 25µl of the vehicle was applied in the defect. The Enamel Matrix Derivate Group (EMD) received 25 µl of Emdogain™. Mesenchymal stem cells (MSC) were collected from the iliac crest of 2 isogenic rats, isolated with Ficoll and cultured in flasks. The cells were separated by flow cytometry to obtain a cell population of CD45-90+ MSC. In the mesenchymal stem cell groups (MSC, MSC-PGA and MSC-EMD) a pellet with 1,2 x 106 cells was applied in each defect, but in MSC-PGA and MSC-EMD groups the pellet was mixed with 25µl of PGA or EMD, respectively. After euthanasia the mandibles were fixed and scanned by computed microtomography apparatus. Bone volume, bone density, trabecular number and thickness were analyzed by SigmaPlot™. The normality of data was checked and ANOVA One-Way test was used, with a significance level of 5%. Results: Bone volume was increased in all groups in all times (p 0.05). In 60 days all groups were similar and had higher volume than PGA (p 0.05) and higher than C, PGA and MSC (p 0.05). MSC-E also had higher number than C and MSC (p 0.05) but statistically larger than C, EMD and PGA (p > 0.05). In 60 days C and EMD had higher thickness than MSC, MSC-PGA and MSC-EMD (p Conclusion: The adhesion of stem cells to EMD resulted in more bone volume in periodontal regeneration defects only in the period of 30 days. However, the bone density and the trabecular number and thickness were either similar or inferior to EMD alone.