The effects of acetylcholine on the spontaneous activity of the AV node of dog hearts were studied by recording transmembrane potentials of its fibers. Action potentials of most nodal fibers were characterized by prominent phase 4 depolarization and a smooth transition from phases 4 to 0. On the isolated AV nodes, acetylcholine at 1.0 microgram/m1 suppressed the rate of phase 4 depolarization and increased the amplitude of the maximum diastolic potential, resulting in a slowing of spontaneous activity. At 2.0 micrograms/m1, spontaneous activity was completely suppressed. In comparison, spontaneous activity of the isolated His bundle was relatively insensitive to the suppressive effect of acetylcholine at the same concentrations. In the AV node-His bundle preparations in which the AV node was the pacemaker, acetylcholine decreased spontaneous activity by suppressing the phase 4 depolarization of the nodal fibers and shifted the pacemaker of the preparation to the His bundle. The findings provide a basis for predicting that under strong vagal influence, the automaticity of the AV node will be suppressed and the pacemaker of the junctional rhythm will be located at the His bundle.