The cholinergic innervation of the rabbit heart was studied in vitro and in vivo. An isolated preparation, which included the combined atria, S-A node, A-V node and bundle of His, was mounted so that microelectrodes could be placed in either the specialized or nonspecialized tissues. Using a roving monopolar electrode, local transmural electrical stimulation of intracardiac nerves (nerve stimulation) was applied at the A-V or the S-A node. At the A-V node it induced hyperpolarization, reduced the duration and amplitude of action potentials recorded from three regions of the A-V node and the His bundle, whether the preparation was spontaneously beating or electrically driven in either the forward or retrograde direction, and blocked membrane excitation in the nodal and nodal-His regions and to downstream structures. The depressant effects of nerve stimulation were augmented by physostigmine and antagonized by atropine. Addition of acetylcholine to the bath reduced the frequency at which the A-V node conducted, but in the concentrations used did not duplicate the effects of nerve stimulation. The behavior of the S-A node is qualitatively similar to that of the A-V node. In the presence of a sinus rhythm, nerve stimulation applied at the A-V node did not affect the activity of the S-A node, and during artificial atrial drive, nerve stimulation of the S-A node did not affect A-V nodal function, indicating that little or no neural communication exists between the two regions in the isolated preparation. Locally released acetylcholine appears to depress excitability of specialized cells in the central node, independently of the direction of propagation.