In the absence of muscle activity, muscle fibers, muscle interstitial cells, and intramuscular nerves display characteristic reactions presumably aimed at restoring a functioning neuromuscular system and avoiding degenerative events. In partially denervated muscle these include proliferation of interstitial cells, followed by nerve sprouting. The same reactions can be induced in intact muscle by elevation of intramuscular insulin-like growth factor (IGF) levels. To determine whether IGFs may participate in the initiation of restorative reactions in inactivated muscle we analyzed the expression of IGF1 and IGF2 mRNA in botulinum toxin-paralyzed and in denervated rat skeletal muscle, and interfered with the activity of IGFs by locally applying the IGF-binding proteins IGF-BP5 and IGF-BP4. To obtain a resolution of the in situ hybridization signals at the cellular level, a nonradioactive method based on digoxigenin-labeled probes was applied. We found that muscle fiber IGF1 mRNA increased rapidly and transiently in inactivated muscle. The time course of this response was similar to that reported for ACh receptor subunits and myogenins. A similar behavior was observed for the corresponding IGF1 protein. The IGF1 response coincided with the transient muscle interstitial cell proliferation reaction. Local application of recombinant IGF-BP5 or IGF- BP4, which are endogenous and specific extracellular ligands of IGFs, prevented the stimulation of interstitial cell proliferation in paralyzed muscle. Our data demonstrate the elevated production of the growth factor IGF1 among early reactions in electrically inactive skeletal muscle fibers and indicate that muscle IGFs are required for the induction of intramuscular interstitial cell proliferation. These findings support the hypothesis that in the neuromuscular system IGF1 plays an important role in initiating cellular reactions in the vicinity of inactive muscle fibers.