Calcium sulfate (CS) can be used as an antibiotically impregnated bone substitute in a variety of clinical constellations. Antibiotically loaded bone substitutes find specific application in orthopedic and trauma surgery to prevent or treat bone infections especially in relation to open bone defects. However, its use as a structural bone graft reveals some concerns due to its fast biodegradation. The addition of calcium carbonate and tripalmitin makes CS formulations more resistant to resorption leaving bone time to form during a prolonged degradation process. The aim of the present study was the evaluation of biocompatibility and degradation properties of newly formulated antibiotically impregnated CS preparations. Three different types of CS bone substitute beads were implanted into the tibial metaphysis of rabbits (CS dihydrate with tripalmitin, containing gentamicin (Group A) or vancomycin (Group B); Group C: tobramycin-loaded CS hemihydrate). Examinations were performed by means of x-ray, micro-computed tomography (micro-CT) and histology after 4, 6, 8 and 12 weeks. Regarding biocompatibility of the formulations, no adverse reactions were observed. Histology showed formation of vital bone cells attached directly to the implanted materials, while no cytotoxic effect in the surrounding of the beads was detected. All CS preparations showed osteogenesis associated to implanted material. Osteoblasts attached directly to the implant surface and revealed osteoid production, osteocytes were found in newly mineralized bone. Group C implants (Osteoset^®) were subject to quick degradation within 4 weeks, after 6-8 weeks there were only minor remnants with little osteogenesis demonstrated by histological investigations. Group A implants (Herafill^®-G) revealed similar degradation within atleast 12 weeks. In contrast, group B implants (CaSO4-V) were still detectable after 12 weeks with the presence of implant-associated osteogenesis atlatest follow-up. In all of these preparations, giant cells were found during implant degradation on surface and inside of resorption lacunae. None of the analyzed CS preparations triggered contact activation. All implants demonstrated excellent biocompatibility, with implants of Group A and B showing excellent features as osteoconductive and -inductive scaffolds able to improve mechanical stability.