Intra-tissue distribution of tetrodotoxin (TTX) in the skin of larva, juvenile, and adult Japanese newt Cynops pyrrhogaster was investigated by means of a monoclonal antibody-based immunoenzymatic technique. In the investigation, TTX was localized at immature glands in juvenile, and at the granular cells composing of granular and mixed glands in adult specimens under a light microscope. No specific stain was recognized in larval section. A duct like structure extending from the granular gland towards super epithelial layer was visualized in the toluidine blue treated skin section of adult newt. When stimuli by wiping with gauze ('handling stimulus') were given, C. pyrrhogaster was found to secrete an applicable amount of TTX and 6-epiTTX from the skin, suggesting that the newt has a granular gland of TTX to secrete it towards the body surface possibly as a biological defensive agent.