Many bacterial toxins act by covalently altering molecular targets within the cytosol of mammalian cells and therefore must transport their catalytic moieties across a membrane. The Protective-Antigen (PA) moiety of anthrax toxin forms multimeric pores that transport the two enzymatic moieties, the Lethal Factor (LF) and the Edema Factor, across the endosomal membrane to the cytosol. The homologous PA-binding domains of these enzymes contain N-terminal segments of highly charged amino acids that are believed to enter the pore and initiate N- to C-terminal translocation. Here we describe a semisynthesis platform that allows chemical control of this segment in LF(N), the PA-binding domain of LF. Semisynthetic LF(N) was prepared in milligram quantities by native chemical ligation of synthetic LF(N)(14-28)alphathioester with recombinant N29C-LF(N)(29-263) and compared with two variants containing alterations in residues 14-28 of the N-terminal region. The properties of the variants in blocking ion conductance through the PA pore and translocating across planar phospholipid bilayers in response to a pH gradient were consistent with current concepts of the mechanism of polypeptide translocation through the pore. The semisynthesis platform thus makes new analytical approaches available to investigate the interaction of the pore with its substrates.