Palytoxin (PTX) is a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter that has potent cardiotoxic properties. In embryonic chick ventricular cells, PTX increased [Ca2+]i (K0.5 = 5 nM) in a manner that was dependent on the presence of extracellular Ca2+. The action of PTX was not consequent to its depolarizing action, to the opening of voltage-dependent Ca2+ channels, to an intracellular Na+ load, or to intracellular acidification. Flow cytometric analysis of the [Ca2+]i distribution in PTX-treated cells showed that only the largest ventricular cells responded to the toxin. All ventricular cells responded to PTX by intracellar acidification. PTX also increased 22Na+ uptake by cardiac cells (K0.5 = 100 nM) via a pathway that was sensitive to 3,4-dichlorobenzamil (K0.5 = 8 microM), suggesting a possible involvement of the Na+/Ca2+ antiporter. We conclude that the action of PTX in chick cardiac cells is distinct from that in erythrocytes or in fibroblasts and that it likely involves several distinct mechanisms. A primary action of PTX could be to open a Ca2+ uptake pathway in the plasma membrane, which would then trigger 22Na+ uptake by the Na+/Ca2+ antiporter.