Satellite cells (quiescent precursors in normal adult skeletal muscle) are activated for growth and regeneration. Signaling by nitric oxide (NO) and hepatocyte growth factor (HGF) during activation has not been examined in a model that can distinguish quiescent from activated satellite cells. We tested the hypothesis that NO and HGF are required to regulate activation using the single‐fiber culture model. In normal fibers, HGF and inhibition of NO synthase (NOS) each increased activation without stretching, and NOS inhibition reduced stretch‐activation. Activation in unstretched mdx and NOS‐I(−/−) fibers was three‐ to fourfold higher than normal, and was reduced by stretching. Distinctions were not due to different pax7‐expressing populations on normal and mdx fibers. The population of c‐met–expressing satellite cells on normal fibers was increased by stretch, demonstrating functional heterogeneity among normal satellite cells. Cycloheximide did not prevent the stretch‐related increase in c‐met expression, suggesting c‐met may be an immediate–early gene in satellite cell activation. Results have important implications for designing new therapies that target the role of exercise in health, aging, and disease. Developmental Dynamics 236:240–250, 2007. © 2006 Wiley‐Liss, Inc.