Several lines of evidence show that transplantation of osteoblastic cells or genetically engineered nonosteogenic cells expressing osteoblastrelated genes into bone defects effectively promotes bone regeneration. To extend this possibility, we investigated whether oral mucosal fibroblasts are capable of differentiating into osteoblastic cells by conducting in vitro and in vivo experiments. We investigated the effects of bone morphogenetic protein-2 (BMP-2) on osteoblast differentiation of cultured fibroblasts isolated from canine buccal mucosa. We also transplanted green fluorescence protein (GFP)-expressing fibroblasts with gelatin/BMP-2 complexes into the subfascial regions of athymic mice, and investigated the localization of GFP-positive cells in the ectopically formed bones. The cultured canine buccal mucosal fibroblasts differentiated into osteoblastic cells by increasing their alkaline phosphatase (ALP) activity and Osteocalcin, Runx2, and Osterix mRNA expression levels in response to BMP-2. Transplantation experiments of GFP-expressing oral mucosal fibroblasts with gelatin/BMP-2 complexes revealed that 17.1% of the GFP-positive fibroblasts differentiated into ALP-positive cells, and these cells accounted for 6.2% of total ALP-positive cells in the ectopically formed bone. This study suggests that oral mucosal fibroblasts can differentiate into osteogenic cells in response to BMP-2. Thus, these cells are potential candidates for cell-mediated bone regeneration therapy in dentistry. Anat Rec, 295:1327Rec, 295: -1335