Hemorrhagic toxin (toxin HT) was purified from Clostridium sordellii culture filtrate. The purification steps included ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography, using a monoclonal antibody to toxin A of Clostridium difficile as the ligand. Toxin HT migrated as a major band with a molecular weight of 525,000 and a minor band at 450,000 on nondenaturing gradient polyacrylamide gel electrophoresis. The molecular weight was estimated at 300,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing indicated an apparent pI of 6.1. Toxin HT was cytotoxic for cultured cells and lethal for mice by intraperitoneal injection, and it elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Immunodiffusion analysis revealed a reaction of partial identity between toxins A and HT. Immunological cross-reactivity between these toxins was further demonstrated by immunoblotting and by neutralization of toxin HT biological activity with antibodies to toxin A. A sensitive indirect enzyme-linked immunosorbent assay was used to examine the affinity involved in homologous and heterologous antigen-antibody interactions. Our findings show that toxin HT has biological activities and immunological properties similar to those of toxin A; however, the toxins are not identical.