Osteocytes are extremely sensitive to fluid shear stress, a phenomenon that may be related to mechanical adaptation of bone (FASEB J 9:441,1995). Here we examined the effect of pulsating fluid flow (PFF, 0.5±0.02 Pa, 5 Hz, 0.4 Pa/sec) on the release of NO, in relation with upregulation of prostaglandin E2 (PGE₂). Chicken calvarial osteocytes, but not periosteal fibroblasts, as well as mouse calvarial cells responded to PFF with a rapid and transient 2 to 3-fold stimulation of NO release. The effect was maximal after 5 min and leveled off thereafter. PFF also stimulated PGE₂ release. This effect was significant after 10 min and continued throughout 60 min PFF treatment. Inhibition of NO release bq NG-monomcthyl-L-arginine prevented the effect of PFF on NO as well as PGE₂ release. These results suggest that NO is a mediator of mechanical effects in bone, leading to enhanced PGE₂ release. They further strengthen the hypothesis that fluid flow through the osteocyte canalicular network provides the physical stimulus for mechanosensation in bone.