We tested the ability of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase (NOS), to suppress the osteogenic response in a recently developed model of mechanically induced osteogenesis. L-NMMA was given either as a single intraperitoneal dose, 15 min before the episode of mechanical stimulation, or as four doses every 6 h, commencing 2 h after loading. Administration of L-NMMA before loading completely prevented the increase in cancellous bone formation by mechanical stimulation. This suppression was largely lost when L-NMMA was administered after loading. Thus the response is likely to be due to activation of a preexistent constitutive NOS in bone cells during or very soon after mechanical stimulation. Suppression of the osteogenic response by L-NMMA was prevented by coadministration of L-arginine but not by the inactive isomer, D-arginine. These changes in cancellous bone were mirrored by similar changes on the corticoendosteal and periosteal bone surfaces. These data suggest that early release of NO is a key signal in the transduction of mechanical stimuli into subsequent bone formation.