Hemocytes from the ascidian, Ciona intestinalis, are capable of lysing erythrocytes in vitro following cell membrane contact. With the aim of examining the mechanism of cytotoxicity, we performed inhibition experiments with lipid components of erythrocyte membranes. Cholesterol is not an inhibitor, whereas, among the phospholipids tested, (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine) sphingomyelin inhibits the hemolytic activity of hemocytes. However, thin layer chromatography showed that sphingomyelinase activity was not contained in the chloroform-methanol extracts from hemocyte debris. The inhibition capacity of the components ceramide and phosphorylcholine suggests that the entire sphingomyelin molecule is involved in binding cytolysins. The lysin-lipid interactions probably cause changes in erythrocyte membrane permeability, leading to lysis.