During early stages of embryonic development, the motoneurones of the spinal cord send out axons that penetrate the differentiating muscle masses and establish connections with individual muscle fibres. It has been proposed that during this period the survival, differentiation and axon outgrowth of the motoneurones depend upon retrograde factors produced by the muscles, and in a previous study, we used a quantitative assay for neurite outgrowth from dissociated embryonic spinal neurones in vitro to characterize a neurite-promoting activity in media conditioned by embryonic muscle cells. At the adult neuromuscular junction, if some of the axons supplying a muscle are experimentally interrupted, fine nerve processes 'sprout' from the remaining intramuscular nerves and grow to innervate the denervated muscle fibres. In this situation also, it has been postulated that the denervated fibres release a diffusible sprouting stimulus. Using the same in vitro assay as before, we now report a striking increase of neurite-promoting activity in extracts of neonatal chick leg muscle after total denervation.