Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 +/- 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-kappaB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.