Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.