In the central nervous system of the earthworm, sensory and motor neurons have direct synapses on three giant fibers. To determine the locations of synapses and neural network activated by mechanical stimuli, we optically monitored the activity-dependent staining in the earthworm ventral nerve cord with a styryl dye, N-(3-triethylammoniumpropyl)-4- (4-(dibutylamino)styryl)pyridinium dibromide (FM1-43), and a confocal laser scanning microscope. When scratch stimulus was applied to the body wall of the earthworm, bright fluorescent spots with 3-10 microns in diameter localized only in the stimulated segmental ganglion of the ventral nerve cord. The fluorescent intensity of these spots decreased during dye-free high K+ saline incubation. These results suggest that FM1-43 is useful for activity-dependent staining of invertebrate neurons and their synaptic regions as well as vertebrate nervous system.