Osteoblasts in culture respond to mechanical strains. Fluid flow has been shown to increase intracellular adenosine 3',5'-cyclic monophosphate levels in cultured osteoblasts, and this response is mediated by prostaglandin synthesis. The signal transduction pathway of these cells exposed to fluid flow is still unknown. In the present study, we have demonstrated a 9- and 20-fold increase in the rate of prostaglandin E₂ (PGE₂) production in osteoblasts exposed to low (6 dyn/cm²) and high (24 dyn/cm²) steady shear, respectively. We further observed that fluid flow induced increases in the intracellular levels of inositol trisphosphate (IP₃), another important second messenger. A shear stress of 24 dyn/cm² increased IP₃ levels dramatically for up to 2 h. Removal of flow resulted in a gradual return of IP₃ to basal levels. The stimulation of IP₃ levels was partially inhibited by 20 microM ibuprofen and 14 microM indomethacin, indicating that the IP₃ response was partly dependent on flow-induced prostaglandin synthesis. The IP₃ response was unaffected by daltroban, a specific thromboxane antagonist. These results show that fluid flow induced prostaglandin E₂ production and increased intracellular levels of IP₃ in osteoblasts. This suggests that flow may be the external signal produced by loading and that these messengers may be involved in the transduction of mechanical strain into a biochemical response.