Bone morphogenetic proteins (BMPs) induce osteoblastic responses in cultures of pluripotent mesenchymal cells. The effects of chronic treatment of these cells with BMPs and of withdrawal following exposure, however, have not been fully elucidated. Thus, the aim of this study was to obtain information about the duration of exposure to recombinant human BMP-2 (rhBMP-2) required for expression and retention of osteoblastic characteristics with subsequent formation of a mineralized extracellular matrix in mesenchymal cell cultures. C3H10T1/2 cells and bone marrow stromal cells were cultured with 1 mg/ml rhBMP-2 for either 0, 7, 14, 21, or 28 days, with the remainder of the 4 week total culture period in the absence of rhBMP-2. Growth and expression of osteoblastic characteristics were examined at the end of each week. C3H10T1/2 cells responded to increasing duration of exposure to rhBMP-2 with increased cell growth. Additionally, the longer the cells were exposed to rhBMP-2, the more fully they expressed and sustained osteoblastic traits, i.e., they exhibited duration of exposure-dependent higher levels of alkaline phosphatase and osteocalcin and larger total amounts of mineral in the matrix. In comparison, exposure of bone marrow stromal cells to rhBMP-2 for at least 14 days restrained cell growth and prevented detachment. With respect to osteoblastic traits, stromal cells exposed to rhBMP-2 also exhibited a dependence on the duration of exposure, however, cultures treated for 14, 21, or 28 days exhibited similar levels of alkaline phosphatase activity and comparable amounts of calcium in the mineralizing matrix.