Mechanical loading alters the metabolism of articular cartilage, possibly due to effects of shear stress on chondrocytes. In cultured chondrocytes, glycosaminoglycan synthesis increases in response to fluid-induced shear. This study tested the hypothesis that shear stress increases nitric oxide production in chondrocytes, and nitric oxide then influences glycosaminoglycan metabolism. Inhibitors of nitric oxide synthase, G proteins, phospholipase C, potassium channels, and calcium channels were also analyzed for effects on nitric oxide release and glycosaminoglycan synthesis. Fluid-induced shear was applied to primary high-density monolayer cultures of adult bovine articular chondrocytes using a cone viscometer. Nitric oxide release in chondrocytes increased in response to the duration and the magnitude of the fluid-induced shear. Shear-induced nitric oxide production was reduced in the presence of nitric oxide synthase inhibitors-but was unaffected by pertussis toxin, neomycin, tetraethyl ammonium chloride, or verapamil. The increase in glycosaminoglycan synthesis in response to shear stress was blocked by nitric oxide synthase inhibitors, pertussis toxin, and neomycin but not by tetraethyl ammonium chloride or verapamil. The phospholipase C inhibitor, neomycin, also decreased glycosaminoglycan synthesis in the absence of flow-induced shear. As studied here, shear stress increased nitric oxide production by chondrocytes, and the shear-induced change in matrix macromolecule metabolism was influenced by nitric oxide synthesis, G protein activation, and phospholipase C activation.