The mode of depolarizing action of palytoxin (PTX) was compared with that of grayanotoxin-I (GTX-I) to examine the site of action of PTX in cardiac tissues isolated from dogs and guinea pigs: PTX at above 1 X 10(-10) mol/l depolarized the membrane of canine Purkinje fibres, canine and guinea-pig ventricular muscles regardless of stimulation. The PTX-induced depolarization was resistant to 1 X 10(-5) mol/l tetrodotoxin (TTX) but was attenuated by low Na medium. GTX-I at 1 X 10(-5) mol/l depolarized the membrane of ventricular muscles from dogs and guinea pigs only when they were stimulated. Although GTX-I caused depolarization in Purkinje fibres in both stimulated and rested conditions, the effect was greater in stimulated fibres. In the presence of GTX-I, ventricular muscles generated long-lasting action potentials as a response to stimuli. TTX antagonized the GTX-I-action. After treatment of PTX, GTX-I still induced the long-lasting action potential and TTX could antagonize it. Ouabain at 1 X 10(-6) or 3 X 10(-6) mol/l partially inhibited the PTX-induced depolarization in guinea-pig papillary muscles. The data suggest that the TTX-sensitive Na channel is intact after treatment with PTX and that PTX acts on a site different from the Na channel, possibly on the Na+, K+-ATPase in cardiac cells.