In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins.