The production of neurotrophin-4 (NT-4) in rat skeletal muscle was found to depend on muscle activity. The amounts of NT-4 messenger RNA present decreased after blockade of neuromuscular transmission with α-bungarotoxin and increased during postnatal development and after electrical stimulation in a dose-dependent manner. NT-4 immunoreactivity was detected in slow, type I muscle fibers. Intramuscular administration of NT-4 induced sprouting of intact adult motor nerves. Thus, muscle-derived NT-4 acted as an activity-dependent neurotrophic signal for growth and remodeling of adult motor neuron innervation. NT-4 may thus be partly responsible for the effects of exercise and electrical stimulation on neuromuscular performance.