This paper reports the effects of drugs affecting the homeostasis of cytosolic-free calcium on in vitro yeast phagocytosis by hemocytes of the colonial ascidian Botryllus schlosseri. Significant inhibition of phagocytosis is observed after exposure of hemocytes to 10 microM or higher concentrations of thimerosal, which is known to deplete intracellular calcium stores in mammalian cells. The two calcium channel blockers nifedipine and verapamil significantly decrease the phagocytic index, the minimum effective concentrations being 10 and 50 microM, respectively. As these substances have no effects at lower concentrations, they probably act through the inhibition of Ca(2+)-ATPase activity, required to restock intracellular calcium stores, due to their interaction with calmodulin. Analogously, pimozide, which suppresses ATPase activity by interacting with calmodulin, and thapsigargin, which inhibits Ca(2+)-ATPase activity, significantly reduce the phagocytic index. Moreover, nifedipine, by altering cytosolic calcium homeostasis, also lowers the production of superoxide anion associated with phagocytosis. Results indicate that in ascidians, as in mammals, a rise in intracellular calcium is required for phagocyte activation and induction of the respiratory burst.