Loading-induced flow of fluid is a signal for bone cell adaptive responses, but the nature of the flow-derived stimulus which activates the cell is debated. Candidate stimuli include shear stress, streaming potentials and chemotransport. In this study the nature of the cell stimulus was addressed by varying the shear stress, using nitric oxide (NO) and prostaglandin E2 (PGE2) production as a parameter of bone cell activation. Mouse bone cell cultures were treated for 15 minutes with or without pulsating fluid flow (PFF). In a few experiments, dextran was added to the fluid to increase the shear stress without affecting streaming potentials or chemotransport. NO and PGE2 production were dose-dependently stimulated by PFF. Application of dextran in the flow medium enhanced both NO and PGE2 production by bone cells. It was demonstrated that the production of NO and PGE2 by bone cells is enhanced by fluid flow of increasing shear stress. Therefore, the stimulus leading to NO and PGE2 production is shear stress rather than streaming potentials or chemotransport.