The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 – residues 402–423, H2 – 437–457) linked by a hydrophilic loop (PMT-TL – 424–436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix–loop–helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.