Perfusion culture of osteoprogenitor cells seeded within porous scaffolds suitable for bone tissue engineering is known to enhance deposition of a bone-like extracellular matrix, and the underlying mechanism is thought to involve flow-induced activation of mechanotransductive signaling pathways. Basic studies have shown that mechanotransduction is enhanced by impulse flow and may be mediated through autocrine signaling pathways. To test this, an intermittent flow regimen (5 min on/5 min off ) that exerts impulses on adherent cells and permits accumulation of secreted factors in the cell microenvironment was compared to continuous flow for its ability to stimulate phosphorylation of ERK and p38, synthesis of prostaglandin E2 (PGE2), and expression of mRNA for collagen 1alpha1 (Col-1alpha1), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). Studies were performed using bone marrow stromal cells cultured in osteogenic media, and parallel-plate flow chambers were used to exert a shear stress of 2.3 dyn/cm2 on cell layers. Results show that continuous flow significantly enhanced phosphorylation of ERK and p38 after 30 min relative to intermittent flow, while intermittent flow significantly increased accumulation of PGE2 in the circulating medium by 24 h relative to continuous flow. Neither continuous nor intermittent flow affected mRNA expression of Col-1alpha1 and OPN after 4 h, but when monolayers were stimulated for 24 h and then allowed to differentiate under static conditions for an additional 13 days, expression of Col-1alpha1, OPN, BSP, and OCN under continuous and intermittent flow was similar and significantly elevated relative to static controls. This study demonstrates that the variation of perfusion regimen modulates mechanotransductive signaling.