Calcium sulfate (CaS) has been shown to be a reasonable alternative to autogenous bone graft for treating bone lesions in dentistry. The aim of this work was an histological study of the bone healing of defects treated with calcium sulfate in the form of cement or beads, in animal. Eight New Zealand rabbits, weighing about 2.5 Kg were used in this study. In each rabbit, four 6 mm bone defects were created in the tibial metaphysis. The 2 defects in the right tibia were filled with calcium sulfate as cement, while the 2 defects in the left one were filled with calcium sulfate as beads. Four rabbits were killed after respectively 2 and 4 weeks, with an intravenous injection of Tanax, and the block sections, containing the bone defects, were retrieved. A total of 16 defects filled by cement and a total of 16 defects filled by beads were retrieved. The specimens were processed to obtain thin ground sections with the Precise 1 Automated System. In the first phases of healing it was possible to observe an intense osteoblastic activity, and in some areas osteoid matrix was present. After two weeks the calcium sulfate (both cement and beads) was still present, and biological fluids and cells were present inside the material. Newly formed bone surrounded the calcium sulfate and filled about 10% of the defect. After four weeks the calcium sulfate was almost completely resorbed and substituted by new bone. Approximately 34% of the defects were filled by newly formed bone. BEI and XRM evaluations showed the structural components of the filled defects. In none of the specimens were inflammatory cells present. No significant differences were found using both calcium sulfate as cement and beads, and they both have shown a high biocompatibility, appearing to promote newly bone formation in the rabbit model, and they did not induce any untoward effect on the bone regeneration processes.