Following injury to a peripheral nerve the denervated distal nerve segment undergoes remarkable changes including loss of the blood-nerve barrier, Schwann cell proliferation, macrophage invasion, and the production of many cytokines and neurotrophic factors. The aggregate consequence of such changes is that the denervated nerve becomes a permissive and even preferred target for regenerating axons from the proximal nerve segment. The possible role that an original end-organ target (e.g. muscle) may play in this phenomenon during the regeneration period is largely unexplored. We used the rat femoral nerve as an in vivo model to begin to address this question. We also examined the effects of disrupting communication with muscle in terms of accuracy of regenerating motor neurons as judged by their ability to correctly project to their original terminal nerve branch. Our results demonstrate that the accuracy of regenerating motor neurons is dependent upon the denervated nerve segment remaining in uninterrupted continuity with muscle. We hypothesized that this influence of muscle on the denervated nerve might be via diffusion-driven movement of biomolecules or the active axonal transport that continues in severed axons for several days in the rat, so we devised experiments to separate these two possibilities. Our data show that disrupting ongoing diffusion-driven movement in a denervated nerve significantly reduces the accuracy of regenerating motor neurons.