Biochemical and immunologic properties of the cytotoxin (leukotoxin) produced by Pasteurella haemolytica were examined. Crude, bacteria-free supernatants from logarithmic phase P haemolytica were fractionated, using a series of column chromatographic techniques. Sequential anion exchange chromatography, gel-filtration chromatography, and chromatofocusing resulted in a cytotoxic substance (cytotoxin-C) of approximately 160 kilodaltons (kD), as determined by use of gel-filtration chromatography. Polyacrylamide-gel electrophoresis of cytotoxin-C yielded 3 protein bands with relative mobilities of 0.37, 0.42, and 0.63. On the basis of immunoblotting with a cytotoxin-neutralizing bovine immunoglobulin for antigen detection, the 2 low-mobility bands shared a strong region of immunogenicity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, principal protein constituents of cytotoxin-C were found at 160, 66, 57, and 23 kD. Using immunoblotting with cytotoxin-neutralizing immunoglobulin, strong, distinct reactions with the 66- and 57-kD bands were detected. Immunization of rabbits and mice with cytotoxin-C resulted in sera that reacted strongly with cytotoxin-C in enzyme-linked immunosorbent assays and immunodiffusion assays. The major immunogenic proteins also were detected by use of immunoblotting with anticytotoxin-C sera from rabbits and mice. Postinoculation rabbit sera neutralized crude cytotoxin.