Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in the presence of osteogenic factors (dexamethasone, beta-glycerophosphate, and ascorbate) were stimulated repeatedly (every second day) with shearing flow (1.6 dyn/cm(2)) for 5, 30, or 120 min, and assayed for systematic changes in cell number and phenotypic markers of osteoblastic differentiation. Cells exposed to fluid flow on days 2 and 4 after the addition of osteogenic factors and assayed at day 6 exhibited a modest decrease in cell number and increase in normalized alkaline phosphatase activity, suggesting the detachment of a non-osteogenic subpopulation. Cells exposed to fluid flow on days 6, 8, 10, and 12 and assayed at day 20 demonstrated maximal expression of osteopontin and bone sialoprotein mRNA with 30 min duration of flow. Concurrently, at day 20 expression of the adipogenic marker, lipoprotein lipase, was minimal with a 120-min duration of flow. These results indicate that repeated application of shear stress stimulates late phenotypic markers of osteoblastic differentiation of BMSCs in a manner that depends on the duration of stimulus. Finally, accumulation of prostaglandin E(2) in culture medium in response to shearing flow systematically decreased with repeated exposure to 30 and 120 min of shear stress (from day 6 to day 12), suggesting an adaptation of the cells to fluid flow.