Microelectrode techniques were used to study the electrophysiological properties of pacemaker fibers in isolated atrioventricular valves of the rabbit heart. In spontaneously beating tricuspid and mitral valve preparations superfused with normal Tyrode solution, leading pacemaker activity was generated by fibers located on the valve leaflet. Impulse conduction from the automatic focus to atrial myocardium was slow, and in approximately 50% of preparations, exit block occurred. Valve pacemaker fibers had significantly different action potential characteristics compared with the primary pacemaker cells of the sinoatrial node. However, the activity of both pacemaker types was largely insensitive to tetrodotoxin but readily suppressed by verapamil. Endogenous neurotransmitters, released from postganglionic nerve endings in the vicinity of valve pacemaker fibers, exerted profound chrono- and dromotropic effects. Brief cholinergic stimuli strongly influenced valve pacemaker cycle length in a phase-dependent manner, qualitatively similar to that in the sinoatrial node. During overdrive of valve pacemaker fibers with external bipolar stimuli, locally released neurotransmitters modified automaticity and conduction which, under certain conditions, favored pacemaker escape. These data demonstrate that the atrioventricular valve leaflets contain cardiac fibers capable of generating spontaneous impulses and that intrinsic autonomic nerves may play a role in precipitating premature extrasystoles.