Spore coat proteins of several strains of Clostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent-) strain of this organism. The results indicated that spore coat proteins from many strains of C. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulation-specific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393-410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.