Bacillus subtilis subtilisin is predicted to be synthesized as a preproenzyme according to the sequence analysis of its gene. We have synthesized the [35S] methionine-labeled preprosubtilisin in vitro and processed the precursor to prosubtilisin by the addition of membrane vesicles derived from vegetative cells of B. subtilis and Triton X-100. Radiosequencing of the prosubtilisin allowed the precise determination of the signal peptidase cleavage site. The preprosubtilisin was found to have a 29-amino-acid-long signal peptide with the signal peptidase cleavage sequence of AlaGln-AlaAla. Fusion of the signal peptide sequence to the mature TEM beta-lactamase structural gene allowed the production of an active and secreted form of beta-lactamase in vivo. An N-terminal sequence analysis of this product indicated that the observed in vivo signal peptidase cleavage site was exactly the same as that determined by in vitro analysis. During the development of the in vitro processing system, we demonstrated that the replacement of the subtilisin transcription regulatory sequence by a vegetative promoter allowed the vegetative expression and secretion of subtilisin. Thus, the late expression of the native subtilisin gene is mainly controlled at the transcription level and the secretion/processing systems are available for vegetative production of subtilisin.