Objectives: This study examined the role of the fibrin sealant Tisseel during early bone formation in rat skull defects. Methods: Forty-eight rats were randomly assigned to four groups, and each group was further divided into two subgroups, which were examined at 4 and 8 weeks after implantation. The defect was filled in different manners: Group 1, no graft; Group 2, tooth ash-plaster graft; Group 3, Tisseel and tooth ash-plaster graft; Group 4, ovariectomy and tooth ash-plaster graft; and Group 5, ovariectomy, Tisseel, and tooth ash-plaster graft. Histologic sections were obtained for histomorphometric analysis of the defects at 4 and 8 weeks after surgery. Results: Overall, there was a significant difference (p = 0.000) in new bone formation among the 4-week groups. At 4 weeks, there were significant differences between groups 1 and 2 (p = 0.004), 1 and 3 (p = 0.004), 1 and 4 (p = 0.004), 2 and 4 (p = 0.004), 2 and 5 (p = 0.004), 3 and 4 (p = 0.045), and 4 and 5 (p = 0.013). Conclusions: Significantly less new bone was formed with ash alone after ovarian resection or with ash and Tisseel combined compared with grafting without ovarian resection. New bone formation was significantly better when ash was used alone or ash and Tisseel were combined.