Choline acetyltransferase activity was estimated in hearts of dogs, cats, rabbits and rats. The ventricles, which form about 90 per cent of the fresh weight of the hearts, are responsible for about two thirds of the total enzyme activity of the heart in dogs, cats and rats, and in rabbits for about half the total activity of the heart. The concentration of the enzyme is higher in the atria than in the ventricles. In dogs the apex of the ventricles showed the same enzyme concentration as the basal parts; in cats and rabbits it was somewhat lower in the apex than in the base. The findings are discussed in relation to the controversial question of a parasympathetic innervation of the mammalian heart ventricles.