The American horseshoe crab, Limulus polyphemus, releases l-5 ml of a proteinaceous secretion at the surface of the carapace when challenged by intracardiac injections of bacterial endotoxin (lipopolysaccharide, LPS) administered at 0.1 pg/ml of blood. Female animals were used because LPS injection stimulates males to shed semen, which then contaminates the exudate. Blood volume is estimated as l/3 of the total body weight of the animal. The secretion (exudate) is presumably the product of the hypodermal glands, whose ducts extend from the dermis to the surface of the carapace (1). The cytolytic activity of the secretion was assayed by the lysis of sheep erythrocytes using methods described previously (2). This hemolytic activity was evident at a 1:400 dilution of the crude exudate and was unaffected by chelation of divalent cations with ethylenediaminetetraacetic acid (EDTA) and inhibition of endogenous protease activity with a,-macroglobulin and phenylmethylsulfonylfluoride. Hemolysis appears to be mediated by a protein, because the hemolytic factor is non-dialyzable with a lo-kDa exclusion limit dialysis membrane, is heat labile, is sensitive to treatment at low (pH 2) and high (pH 12) pH, and is partially sensitive to trypsin. Seawater that ran off the surfaces of the unchallenged animal was not hemolytic. Animals that were injected only with the diluent for LPS (pyrrogen-free 3% NaCl) failed to secrete the hemolytic exudate. SDS-polyacrylamide gel electrophoresis (reducing conditions) showed the LPS-elicited exudate to contain major protein bands at 126, 93, 83, 79, 48, 47,26, 23, 17, 16, 15, and 6 kDa and a number of minor bands between 45 and 30 kDa. The pattern of proteins seen in SDSPAGE shows that the exudate contained little or none of the abundant proteins of the plasma (e.g., ol,-macroglobulin, hemocyanin, and C-reactive protein) and also contains no detectable quantities of the suite of proteins secreted by blood cells stimulated to undergo exocytosis by challenge with LPS or with the Ca’*-ionophore, A23 187 (3). The hemolytic activity partitioned by gel exclusion chromatography on Sephadex G-100 resin with an apparent molecular mass of 25-30 kDa. The most active fractions from the G-100 column were enriched in a major protein band seen in SDS-PAGE (reducing conditions) at 47 kDa, a suite of minor bands between 45 and 30 kDa, and a protein band at 23 kDa. The protein responsible for hemolysis failed to bind to Mono Q anion-exchange resin at low salt concentrations (approx. 50 mM NaCl), consistent with the possibility that it is a basic protein. SDS-PAGE (reducing conditions) showed that the active fractions from the Mono Q column contained five prominent proteins as a band at 45 kDa, a triplet of bands at 31 kDa, and a band at 23 kDa.