The precursors of beta-lactamase fusion proteins having the signal peptide of Bacillus subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5(*) (pPBP5(*)-BlaH6) accumulated in B. subtilis cells in the absence of SecA or Ffh. Using the five purified precursors of secretory proteins including the two fusion proteins, B. subtilis Ffh and SecA, we analyzed the protein targeting mechanism of B. subtilis in vitro. B. subtilis SecA recognized the completely translated precursors of secretory proteins to which Ffh also bound. Moreover, B. subtilis SecA-precursor complex formation was enhanced 15-to 30-fold when the precursor and Ffh were incubated first and then SecA was added, but not vice versa. We also found that B. subtilis SecA directly interacted with Ffh in vitro. These results indicate that B. subtilis SecA and Ffh interact to function cooperatively in a protein translocation pathway including other protein factors, and that Ffh, as well as SecB in Escherichia coli, enhances the binding of SecA to presecretory proteins in B. subtilis cells.