Abstract--Immune dysfunction has been reported in hypertensive rats, and circulating levels of ouabain are increased in some experimental models of hypertension. Ouabain is an inhibitor of the Na⁺/K⁺-ATPase capable of diverse effects on cells of the immune system, but its mode of action on these cells is still unknown. The levels of cytoplasmic calcium ions play an important role in cell signaling, and ouabain may induce an increase in intracellular calcium indirectly through the Na⁺/Ca²⁺ exchanger. The current work examined the possibility that this drug could be exerting its effects on thymocytes through calcium mobilization and an increase in the cytosolic calcium concentration. Intracellular calcium was evaluated by using Balb-c mouse thymocytes loaded with FURA-2. Both intracellular and extracellular calcium pools were mobilized by ouabain (3 to 1000 nmol). The influx of extracellular calcium depended on the Na⁺/Ca²⁺ exchanger and on voltage-dependent calcium channels, as it was inhibited by amiloride and benzamil, consistent with the inhibition of the Na⁺/K⁺ pump. In addition, the increase of calcium from intracellular stores was extremely rapid. Furthermore, an increase in cytosolic calcium levels was obtained with the combination of ouabain and thapsigargin, which was greater than that seen with either drug alone. Our data suggest that low concentrations of ouabain may be acting on thymocytes through a mechanism different from the traditional inhibition of the Na⁺/K⁺-ATPase, as the cytosolic calcium rise was partly dependent on the release from intracellular stores.