This study evaluated the efficacy of periodontal tissue regeneration using guided tissue regeneration (GTR) with a mixture of tooth ash and plaster as a bone substitute material in the treatment of class II furcation defects in dogs. GTR was performed at the site of a surgically formed mandibular premolar bifurcation area in dogs. Four adult dogs were used in the animal study. The animals were in a good periodontal state without any systemic disease. A resorbable membrane (Bio-Gide®, Swiss) and bone graft material using a mixture of tooth ash and plaster were used to examine the biological activity. The defect in the control group was filled a Bio-Gide® only. For the experimental group, the defects were filled with a mixture of tooth ash and plaster and covered by a Bio-Gide®. Oral rinsing with 0.12% chlorhexidine was performed twice a day for 2 weeks. In control group, new bone formation was observed adjacent to the preexisting bone. Soft tissue ingrowth was also noted. In experimental group, new lamellar type trabecular bone formation as well as centum and PDL regeneration was observed in the base of the infrabony pocket. More bone regeneration would be expected when GTR is performed using a mixture of human tooth-ash and plaster of Paris as a bone graft material.