We studied the effects of hypoxia on the electrical activity and ultrastructure of pacemaker cells of sinoatrial nodal tissue excised from rabbit heart. The slope of slow diastolic depolarization was markedly reduced within 15-20 minutes after gassing Tyrode's solution with nitrogen, whereas other action potential parameters were not much affected. At 60-90 minutes in the hypoxic solution, the maximum diastolic potential decreased by 13 mV, and the peak potential decreased to −5 mV, but abbreviated action potentials continued to occur for another 30-60 minutes. After 60-90 minutes of hypoxia, reoxygenation did not produce complete recovery of the action potential. In glucose-free solution, action potentials ceased within 40 minutes of hypoxia, but reoxygenation restored electrical activity. A good correlation was found between the duration of hypoxia and the extent of ultrastructural change in sinoatrial nodal cells. After 15-20 minutes of hypoxia, mitochondria showed decreased electron density of their matrix and loss of intramitochondrial particles. The glycogen content was reduced. After 60-90 minutes of hypoxia, further changes in the mitochondria occurred: clearing of the matrix, fragmentation of cristae, rupture of the outer membrane, and formation of myelin figures. Glycogen had almost completely depleted after reoxygenation. These changes were partly reversible except for the glycogen content. Our results provide electrophysiological evidence that cells in the sinoatrial nodal tissue can withstand long exposure to hypoxia, using energy produced by the anaerobic glycolytic pathway. Circ Res 46: 201-213, 1980