When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.