Résumé : Two different lipid-associating domains have previously been identified in diphtheria toxin fragment B: one of the surface type in the N-terminus of B and one of the transverse type in its middle region. We have now determined about 85% of the primary structure of fragment B and show, here, that the middle part of fragment B contains a highly hydrophobic region of 72 amino acid residues (polarity index: 295) which includes the transverse lipid-associating domain. That this domain is actually involved in a process of membrane penetration is suggested by lipid bilayer conductance measurements of the CNBr peptides of fragment B and trypsin treatment of fragment B-multilamellar liposome complexes.