Certain aspects of the acetylcholine hypothesis of cardiac automatidty have been tested in dtro with spontaneously beating cardiac tissue from rabbits, rats, dams, and hagfish. The beat of atria from rabbits and rats may be depressed or excited by acetylcholine, depending upon the state of the tissue. Proguanil and cocaine inhibition of the beat in the rat may be antagonized by acetylcholine so that reversal of the depression occurs. The action of acetylcholine on the hearts of dams was found to be strictly inhibitory. Proguanil and cocaine, in contrast to their action on mammalian atria, exert a stimulatory effect on the heart of the molluscs studied. In fact, cocaine stimulated these hearts when they were inhibited by acetylcholine.Studies on the non-innervated hagtish heart revealed that this tissue is completely insensitive to the action of acetylcholine. Extracts prepared from beating hearts of this species will accelerate hypodynamic hearts of the hagiish as well as of the mussel. An extract of the neurogenic lobster heart was without effect on the hagiish heart. Proguanil was likewise ineffective in concentrations which produced inhibition and excitation in rat and clam hearts respectively.It was concluded that acetylcholine does not play a role in the myogenic automatism of all species, and that another mechanism is responsible is suggested on the basis of results obtained in the hagfish hearts.