Palytoxin is a coral toxin that seriously impairs heart function but its effects on excitation-contraction (e-c) coupling have remained elusive. Therefore, we studied the effects of palytoxin on mechanisms involved in atrial e-c coupling. In field-stimulated cat atrial myocytes, palytoxin caused elevation of diastolic [Ca²⁺]_i, a decrease in [Ca²⁺]_i transient amplitude, and Ca²⁺ alternans followed by [Ca²⁺]_i waves and failures of Ca²⁺ release. The decrease in [Ca²⁺]_i transient amplitude occurred despite high SR Ca²⁺ load. In voltage-clamped myocytes, palytoxin induced a current with a linear current-voltage relationship (reversal potential ~+5 mV) that was blocked by ouabain. Whole-cell Ca²⁺ current and ryanodine receptor Ca²⁺ release channel function remained unaffected by the toxin. However, palytoxin significantly reduced Ca²⁺ pumping of isolated SR vesicles. In current-clamped myocytes stimulated at 1 Hz, palytoxin induced a depolarization of the resting membrane potential that was accompanied by delayed afterdepolarizations. No major changes of action potential configuration were observed. The results demonstrate that palytoxin interferes with the function of the sarcolemmal Na⁺-K⁺ pump and the SR Ca²⁺ pump. The suggested mode of palytoxin toxicity in atrium involves conversion of Na⁺-K⁺ pumps into non-selective cation channels as a primary event followed by depolarization, Na⁺ accumulation, and Ca²⁺ overload which, in turn, causes arrhythmogenic [Ca²⁺]_i waves and delayed afterdepolarizations.