Molecular changes occurring in type E. Cl. botulinum single-chain toxin as the result of treatment with trypsin under different conditions were studied. The intensity of activation of the precursor and the ensuing changes of its molecular structure were found to depend on the pH of the medium. At pH 6.0 complete activation induced by the trypsin treatment of the single-chain toxin coincided with complete break-up of the polypeptide chain, while at pH 5.0 the toxin was completely activated before all its molecules could acquire the double-chain structure. At pH 4.5 no increase in the potency of the toxin was registered even in those cases when break-up of the molecules was as pronounced as by the moment of complete activation of the toxin at pH 5.0. These data suggest that activation is not direct consequence of break-up of the peptide bond responsible for the formation of a double-chain molecule. Trypsin-induced activation seems to be linked with the splitting of some peptide bond in one of the end areas of the molecule.