Mechanical stimulation of bone cells is critical for maintaining bone mass and strength, and a better understanding of how mechanical stimuli are converted into intracellular signals to activate an anabolic program in osteoblasts/cytes is fundamental to improving treatments for osteo-degenerative diseases (1;2). Weight bearing and locomotion stimulate interstitial fluid flow through the bone canalicular system, and the resultant shear stress is thought to be a major mechanism whereby mechanical forces stimulate osteoblast/osteocyte growth and differentiation (1;2). In primary osteoblasts and osteoblast/cyte-like cell lines, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/ΔfosB mRNAs (3); these genes encode transcriptional regulators important for osteoblast proliferation and differentiation, as demonstrated by the phenotypes of mice that over-express or lack these proteins, respectively (4). Fluid shear stress increased osteo¬blast nitric oxide (NO) synthesis, leading to increased cGMP synthesis and activation of cGMP-dependent protein kinases (PKGs). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membrane-permeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression, and cGMP’s effects were enhanced by increased intracellular calcium, as occurs in mechanically-stimulated osteoblasts. In cells transfected with siRNAs specific for membrane-bound PKG II, shear- and cGMP-induced Erk activa¬tion and fos family gene ex¬pression was nearly abolished and could be restored by trans¬ducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. We further demonstrated that PKG II mediates the fluid shear-induced increase in osteoblast proliferation. We have identified a mechanism whereby PKG II activates Erk and induces proliferation in osteoblasts, which includes PKG II regulation of c-Src and cross-talk between NO/cGMP/PKG and integrin signaling. PKG II-null mice show defective osteoblast Src/Erk signaling, and decreased Erk-dependent gene expression in bone; previous studies in NO synthase-deficient mice have demonstrated an important role of NO in osteoblast biology (5). We now establish a central role of NO/cGMP/PKG II signaling in osteoblast mechano¬transduction, and pro¬pose a model whereby NO/cGMP/PKG II-mediated Src and Erk activation and induction of fos family genes play a key role in the osteoblast anabolic response to mechanical stimulation.