Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common cause of spinal canal stenosis and myelopathy in Orientals. OPLL is characterized by heterotopic new bone formation in ligamentous tissue. To investigate the pathogenesis of OPLL, human posterior longitudinal ligament cells were cultured and their in vitro morphological and biochemical characteristics were studied. Cell cultures from control subjects with normal spinal ligaments did not show any osteoblastic properties. In contrast, cell lines (OG1-OG5) obtained from an OPLL patient showed several different phenotypic characteristics for osteoblasts. OG1 cells showed typical osteoblast-like phenotypic characteristics (i.e., in vitro calcification, high alkaline phosphatase [ALP] activity, and elevation of cAMP levels by parathyroid hormone [PTH]). All cell lines (OG1-OG5) responded to PTH and PGE2 by markedly increasing cAMP levels, ALP activities varied among the cell lines. The OG1 and OG2 cells exhibited a high level of ALP activity. Compared with cell lines from the non-ossification group, the activities were higher in the OG3 and OG4 cells, but not significantly in the OG5 cells. Only in the OG3 cells, CT caused an increase in cAMP level and ALP activity, and its stimulatory effects demonstrated that CT had a direct, in vitro action on ligament cells of OPLL patients to stimulate osteoblastic differentiation. It is clear that some cells from ligaments with OPLL had several phenotypes characteristic of osteoblasts, but cells from ligaments without ossification did not show any osteoblastic properties. This observation is considered to be an important clue to understanding the pathophysiology of OPLL.