The long clinical experiences with acupuncture at Tsu-San-Li seem to suggest that the therapeutic effects may be caused by anticholinergic mechanisms. This study was carried out to investigate the mechanism with Wistar strain rats. Electroacupuncture was performed at Tsu-San-Li of the animals under urethane anesthesia for five times on alternate days. On the next day of the fifth stimulation, the salivary gland secretion, heart rate, respiratory rate and pH of gastric juice were measured before and as long as 40 minutes after injection of acetylcholine (ACh). All responses to ACh were inhibited compared with the control animals. It was concluded from these results that the anticholinergic effects of electroacupuncture at Tsu-San-Li in rats might have been mediated by unknown humoral factors.