Transmembrane action potentials were recorded from multiple sites in isolated canine anteromedial left atrial wall preparations with the anterior mitral valve leaflet attached. The preparations were superfused with Tyrode's solution. When the left atrial wall was electrically stimulated, activity propagated into the mitral valve leaflet. Typical atrial action potentials occurred in atrial wall fibers. However, maximum diastolic potential, total action potential amplitude, and rate of depolarization decreased markedly in the atrium overlying the fibrous annulus (junctional region), and repolarization characteristics were altered in this region. Mitral valve muscle fibers demonstrated still lower maximum diastolic potential, action potential amplitude, and rate of depolarization. Electrical excitation of single discrete regions in the mitral valve leaflet did not result in conduction to the atrium; conduction block occurred in the junctional region. However, simultaneous excitation of several mitral valve sites did cause an impulse to propagate to the atrium, but transmembrane action potentials of junctional fibers were characteristically different from those recorded from the same junctional fibers during activation from the atrium. Muscle fibers in the mitral valve leaflet were capable of developing spontaneous diastolic depolarization, which resulted in automatic impulse initiation, when they were exposed to epinephrine or norepinephrine (1 X l(h 7 to 1 X 10-*M) or when they were stretched. Spontaneous diastolic depolarization and automaticity also occurred occasionally without pharmacological or other experimental interventions; moreover, spontaneous activity originating in the mitral valve leaflet could propagate into and activate the atrial wall. Acetylcholine abolished spontaneous activity. These data suggest that the mitral valve could act as a site of ectopic impulse initiation in the left atrium.