We examined the possibility that periodontal ligament (PDL) cells can differentiate into osteoblasts and/or cementoblasts in freshly isolated PDL tissues and in cultured cells derived from PDL. PDL tissues were obtained from the incisor teeth of bovine lower jaws; gingival connective tissues of the same animals were used as controls. Freshly isolated PDL tissues and cultured PDL cells showed an intense alkaline phosphatase (ALPase) activity both histochemically and biochemically. The production of 3′, 5′-cyclic adenosine monophosphate (cAMP) was greatly increased in response to human parathyroid hormone [PTH(1-34)], in both freshly isolated PDL tissues and cultured PDL cells. In contrast, neither ALPase activity nor PTH-dependent cAMP production was detected in gingival connective tissues and cultured gingival fibroblasts. Furthermore, cultured PDL cells synthesized a protein immunologically cross-reactive with bovine bone gla protein (BGP), a highly reliable marker of osteoblastic cells. When 10−8 M lα, 25-dihydroxyvitamin D3 [lα,25(OH)2D3] was added to the PDL cell cultures, the synthesis of the BGP-like protein was increased 2- to 3-fold. The maximal level of the synthesis was obtained 72 h after the addition of lα,25(OH)2D3 Gingival fibroblasts cultured with or without lα,25(OH)2D3 did not produce any appreciable amounts of the BGP-like protein. These results indicate that the PDL cells have phenotypes typical of osteoblasts, indicating that they may differentiate into osteoblasts and/or cementoblasts.