Interstitial fluid flow generated by skeletal loading may be responsible for load-induced bone remodeling. Production of prostaglandin E₂ (PGE₂), a potent mediator of bone remodeling, is augmented in osteoblasts exposed to fluid flow. Exposure to fluid flow resulted in a slight initial increase in PGE₂ production (1–2 hour), followed by a dramatic increase (2–8 hours). The initial phase of only slightly increased PGE₂ production was dependent on substrate availability. H7, a protein kinase C inhibitor, strongly inhibited flow-induced prostaglandin E₂ production at all time points examined without effecting production in stationary cultures. Blocking protein synthesis with cycloheximide resulted in a 56% reduction in long-term flow-induced PGE₂ production. Thus, the later phase appeared to be the result of an increased number of enzymes as well as increased activity of existing enzymes or increased substrate availability. In conclusion, fluid flow increases PGE₂ production in osteoplasts via a protein kinase C-dependent pathway involvingde novo protein synthesis.