Abstract 1. 1. The ganglion of Ciona intestinalis L. was isolated and extracted. Acetylcholine determinations by bioassay on isolated hearts of Venus and Tapes indicated an acetylcholine content of 20–120 μg/g wet weight. The acetylcholine content of the body wall (without tunic) was found to be between 0·01 and 0·2 μg/g wet weight. 2. 2. Unboiled homogenates of Ciona ganglia lose their acetylcholine activity almost completely within 90 min, but the activity remains if an anticholinesterase (eserine) is added initially. 3. 3. Unboiled homogenates of Ciona ganglia prepared in isotonic medium maintain a major amount of their acetylcholine content. This bound acetylcholine is released upon boiling. 4. 4. The muscles of the body wall respond with contraction to acetylcholine. This effect is not potentiated by eserine. 5. 5. A cholinergic motor innervation is likely. The observed low cholinesterase activity explains the slow relaxation of the musculature in vivo.