An enterotoxin with the ability to induce fluid accumulation in rabbit ileal loops, erythema in the skin of guinea pigs, and lethality in mice appears in cell extracts (CE) and culture filtrates (CF) of sporulating cells of some Clostridium perfringens type A strains. All activities in CE and CF were eluted simultaneously from a Sephadex G-200 column. Different elution patterns were obtained for these activities present in CE and CF. Rabbit immune serum against CF and the active CE fractions eliminated the three biological activities in CE and CF. These activities present in CF and CE were not eliminated by any of the known antitoxins present in diagnostic serum against C. perfringens types A, B, C, D, and E. Immunodiffusion studies with immune serum against active CE fractions and CF indicated a precipitin line of identity between CF and CE of NCTC 8798 and other enterotoxin-positive strains but not enterotoxin-negative strains. Disc electrophoresis of active G-200 fractions on 7.0% polyacrylamide gels revealed a single area containing erythemal activity and mouse lethality. Immunodiffusion with acrylamide gels, containing crude fractionated enterotoxin, and immune serum against partially purified enterotoxin revealed a single precipitin band in the same area as the biological activities. Immunoelectrophoresis of CE of enterotoxin-positive and enterotoxin-negative strains also showed one precipitin band which occurred only with enterotoxin-positive strains. These findings suggest that one component is responsible for the biological activities attributed to the enterotoxin.