A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB–NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB–NBD and HlyA1 interact with a K D of approximately 4μM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1–NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB–NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB–NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.