In bone tissue engineering, flow perfusion bioreactors have shown great potential for accelerated production of functional constructs, but bioreactor culture conditions have not been optimized. The goal of this study was to investigate the short-term (1- 49 h) effects of intermittent steady, pulsatile, and oscillatory fluid flow (peak flow rate = 1.0 mL/min) on MC3T3-E1 osteoblast activity within a collagen-glycosaminoglycan scaffold. Bioreactor culture at a continuous low flow rate (0.05 mL/min) was also evaluated. Fluid flow exposure stimulated 8 to 51, 15 to 48, and 1.4 to 2.7 greater cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production, and osteopontin expression, respectively, whereas membrane-associated prostaglandin E synthase-1 was 1.8 greater only under steady flow. Overall, intermittent flow (high flow rate) caused greater stimulation than a continuous low flow rate without a loss in cell number. Pulsatile and oscillatory fluid flow tripled COX-2 expression from 25 to 49 h (p < or =0.04), whereas under steady flow, PGE2 production dropped 52% at 49 h (p = 0.05). These results indicate that intermittent flow is advantageous for mechanically stimulating osteoblasts while maintaining cell viability. In addition, results at 49 h suggest that dynamic (pulsatile and oscillatory) flow may be more stimulatory than steady flow over long-term culture.