The primary aim of the present study was to develop for Bacillus subtilis an in vitro transcription translation processing translocation system, which would allow the identification and the purification of the secretory components involved, and which would enable the characterisation of Bacillus strains with alterations in their secretory machinery. An efficient in vitro transcription and translation system for Bacillus subtilis has been established consisting entirely of this organism's components. Furthermore, an in vitro assay for Bacillus subtilis signal peptidase has been developed, which enabled the primary biochemical characterisation of this enzyme. This method is the first example of such a system for a signal peptidase derived from a Gram positive organism, and it enables the purification of the Bacillus subtilis signal peptidase as well as as a more detailed biochemical characterisation of the protein. In addition, the processing assay allows the characterisation of Bacillus mutants altered in their processing capability.