The potent marine toxin, palytoxin (PTX) binds to the Na⁺,K⁺-ATPase (NKA) and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. The ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺-overload-induced oncotic cell death however, has not been examined. The purpose of the present study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca²⁺]_i that was dependent upon extracellular Ca²⁺. The increase in [Ca²⁺]_i was blocked by the pretreatment with ouabain with an IC50 <1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). Subsequent to the rise in [Ca²⁺]_i, PTX also caused an increase in the uptake of the vital dye, ethidium bromide (EB), but not YO-PRO-1. EB uptake was blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently-expressed GFP (Mr = 27 kDa), and the rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by addition of ouabain after PTX, 2) blocked by the cytoprotective amino acid, glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺-overload.