Morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO) inside their vacuoles. As the release of their vacuolar content at the border of contacting incompatible colonies entails the formation of necrotic masses, which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible colonies, MC degranulate and after 60 min the cytotoxicity index becomes significantly greater than that observed in controls with autologous plasma. The rise in cell mortality is completely inhibited by the addition of the PO inhibitor Na benzoate 10 mM or of the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamidine at 1 mM concentration, although MC regularly degranulate. In the presence of Na benzoate 10 mM there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of nonfusible colonies. These results strongly suggest that PO is the enzyme responsible of the cytotoxicity observed both in hemocyte cultures and rejection reactions.