The translocation (T)-domain plays a key role in the entry of diphtheria toxin into the cell, where it inserts into the endosomal membrane and transfers the catalytic domain into the cytosol in response to endosomal acidification. Protonation of the three acidic residues located in the hydrophobic helical hairpin TH8-TH9 (E349, D352 and E362) has been suggested to modulate transmembrane insertion of the T-domain. Here, we test this hypothesis by combining site-directed mutagenesis with assays that test the conformational switching and bilayer insertion in the context of either isolated helical fragments or that of the entire T-domain. The propensity of individual helices to adopt a transmembrane conformation, studied using translocon-assisted insertion, reveal that only the most hydrophobic helix TH8 has a marginally favorable free energy of insertion. The free energy for TH8-TH9 hairpin was more favorable, yet much lower than that for the entire protein, suggesting a cooperative effect for T-domain membrane insertion. While mutations of acidic residues had no effect on insertion of individual helices, they had an effect in the context of the entire protein. E.g., E362Q mutant labeled with the environment-sensitive fluorophore NBD in the middle of TH9 inserts more efficiently than the WT under mildly acidic conditions, but less efficiently upon further acidification. CD spectroscopy, intrinsic fluorescence and size-exclusion chromatography suggest that this mutant is more susceptible to acid destabilization and is prone to aggregation at neutral and mildly acidic pH. We suggest that the principal role of acidic residues in TH8-TH9 segment is not to modulate pH-dependent insertion directly, but to control the early stages of refolding in solution by protecting hydrophobic surfaces of the protein prior to initiation of membrane interactions. Supported by NIH GM-069783, Fulbright-CONICYT.