To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E₂(PGE₂). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF;​ 0.7 ± 0.03 Pa at 5 Hz), and PGE₂ release was measured. Blockers of Ca²⁺+ entry into the cell or Ca²⁺+ release from internal stores markedly inhibited the PFF-induced PGE₂ release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca²⁺+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE₂ release. These results are consistent with the hypothesis that PFF raises intracellular Ca²⁺+ by an enhanced entry through mechanosensitive ion channels in combination with Ca²⁺+- and inositol trisphosphate (the product of phospholipase C)-induced Ca²⁺+ release from intracellular stores. Ca²⁺+ and protein kinase C then stimulate phospholipase A₂ activity, arachidonic acid production, and ultimately PGE₂ release.