Culture of osteoblast is extremely valuable in analyzing biological features that are specific to bone. Authors tried isolation and primary culture of osteoblast by sequential digestion of newborn rat calvariae with enzyme solution containing collagenase and trypsin. Culture and successive subculture were carried out on DMEM-FCS media. To characterize the isolated bone cells, morphological features in culture media were determined and alkaline phosphatase activity were measured. Immediately after extraction from the calvariae, all of the cells were small round bodies and differed little from one another on inverted microscope. Upon culture, cells became clustered and have polygonal shape with stellate or dendritic borders in the centers of clusters. Expression of high alkaline phosphatase activity was maintained until termination of primary culture at 2 weeks (77-729IU/L/mg of protein) in contrast to undetectable activities of human mono-and lymphocyte as a control. On subculture, morphology of the cells were well maintained but alkaline phosphatase activity was relatively low as compared with original culture cells.