SUMMARY Clostridium perfringens type-A α-toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a pH 4·6 gradient. Milligram yields of highly purified α-toxin with 10 to 15% recovery of activity were obtained in single fractions. Two forms of α-toxin were identified by electrofocusing. The major peak of activity possessed a pi of 5·49±0·06(αA) and the minor, a pi of 5·25(αB). The former was free from detectable collagenase (pi 4·54), hyaluronidase (pi 4·73), θ-toxin (pl 6·56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with αA in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of αA. Fractions αA and αB both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of α-toxin in the presence of 6m urea resulted in the detection of only one component, αurea, with a pi identical to αA. It also possessed all three biological activities of α-toxin. Removal of the urea and refocusing was accompanied by the reappearance of αB. The occurrence and formation of αB could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that αA and αB could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.