Cholinergic substance was found in the pars nervosa of the bovine neurohpophysis, the caudal neurosecretory storage organ (urohypophysis) of marina and fresh water teleosts and the brain of the crayfish, according to the method described by Welsh with the isolated heart of Schizothaerus nuttalli (1943). The extract of bovine neural lobe, obtained by boiling the homogenate in acid, was treated with M/100 sodium thioglycolate in order to inactivate the neurohyphysial hormones therein, since the effect of neurohypophysial hormones on the heart beat of Schizothaerus was found to be antagonistic to that of acetylcholine. The extract of bovine neural lobe after this treatment contained the cholinergic substance. In the brain of the crayfish, Procambarus clarkii, the cholinergic substance was detected only in free form. The amount of total cholinergic substance in the brain of the crayfish kept in continuous illumination for 14 to 30 days was equivalent to 44±5.0μg acetylcholine chloride per gram tissue, and that of crayfish kept in the dark for the same periods was equivalent to 47±3.4μg per gram tissue. There is no significant difference between these two values. However, when the amounts are expressed as an equivalent amount of acetylcholine chloride per brain of one animal, they are significantly different (0.19±0.017μg per brain in light adapted animals and 0.27±0.015μg per brain in dark adapted animals). The application of the frog bladder assay method designed for neurohypophysial hormones to an extract of carp urohypophyses revealed that the urohypophysis did not contain detectable amount of neurohypophysial hormones (Fig. 3). In the carp urohypophysis the ratio of free to total cholinergic substance was approximately 1:2. The concentration of total cholinergic substance in the carp urohypophysis was 100 to 150 times that in its brain. The results of the incubation experiment of the carp urohypophysis homogenate with or without eseri ne indicated the presence of cholinesterase activity in the urohypophysis. The amount of cholinergic substance in the urohypophysis of fresh water teleosts was equivalent to 270±59μg acetylcholine chloride per gram tissue in the carp, Cyprinus carpio, 170±33 in the crucian carp, Carassius auratus, and 190 and 120 in the gold fish, Carassius auratus. In the marine teleosts, it was 33, 19 and 21μg per gram tissue in Hexagrammos otakii, Lateolabrax japonicus and Mylio macrocephalus, respetively. The substance is resistant to acids but not to alkalis. Eserine inhibited the action of the substance to the bivalve heart. We have reported for the pars nervosa and the median eminence of various vertebrates and for the urohypophysis of fish that the endings of the neurosecretory axons contain synaptic vesicles in addition to neurosecretory granules. It was true also for the crayfish brain. It contains neurosecretory axons containing synaptic vesicles and neurosecretory granules (Figs. 1 and 2). It is strongly suggested by the present investigation that the cholinergic substance found in the neurosecretory storoge-release organ is acetylcholine and localized in the synaptic vesicles. The role of acetylcholine in the secretion mechanism of the neurosecretory substance was discussed in this connection. This investigation was supported by Grant No. A3678 to H. Kobayashi from the U. S. Public Health Service.