A series of four antibodies against rat osteoblasts have been produced using the hybridoma technique. After bone cells isolated from newborn rat calvariae by a sequential digestion procedure were cultured for 3 days, the cells were trypsinized and further maintained in rotation cultures overnight. Out of the cultured bone cells alkaline phosphatase-positive cells were sorted by flow cytometry and used as immunogens. The clones secreting the antibodies were selected on the basis of the abilities of these antibodies to bind to the bone cells but not to fibroblasts from neonatal rat head skins, in an enzyme-linked immunosorbent assay. Clones of two hybridomas, designated AOB-1 and AOB-2, were used to characterize the antigenic determinant(s) in osteogenic cells. The antibody showed the reactivity with isolated alkaline phosphatase-positive cells, osteogenic tissue cells in newborn rat calvaria, and mandibula, but not with the cells in head skin, lung, kidney, liver, or stomach as determined by immunofluorescence study. Western blot analysis has identified the antigenic determinants possessing apparent molecular weights of 210,000, 110,000, 65,000, 58,000, 40,000, 36,000, 32,000, 28,000, 25,000, 17,000, and 15,000 of osteoblast-rich monolayer cultured cells. According to the cell surface detection with biotin-avidin protein blotting technique, these fractions appear to be present as components of the cell surface of the osteoblast.