Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca2+ concentration ([Ca2+]i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca2+ indicators, fura-PE3 or fluo-3. PTX (10 pM-300 nM) irreversibly increased endothelial [Ca2+]i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca2+]i. Imaging of [Ca2+]i with a confocal microscope revealed that PTX increased [Ca2+]i in all endothelial cells studied (n = 13). An inorganic Ca2+ entry blocker, La3+ (30 microM), had no effect on the increase in [Ca2+]i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca2+]i induced by ATP or thapsigargin. The PTX-induced increase in [Ca2+]i was partially inhibited by ouabain and was abolished by removal of external Ca2+ although decrease of Na+ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1 microM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10 nM calyculin-A had no effect on the increase in [Ca2+]i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca2+]i induced by ATP or thapsigargin. Mn2+ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca2+]i in the endothelium of the rabbit aortic valve by increasing Ca2+ influx through a pathway which is different from that activated by ATP or thapsigargin.