Aqualysin I is a subtilisin-type serine protease secreted into the culture medium by Thermus aquaticus YT-1. It is first produced as a large precursor that consists of a signal peptide, an N-terminal pro-domain, the mature protease domain and a C-terminal pro-domain. To investigate whether the N-terminal pro-domain supplied in trans as an independent peptide plays an important role in the folding and secretion of the protease, the N-terminal pro-domain in E. coli has been expressed independent of the mature domain with or without the C-terminal pro-domain using an expression system with separate promoters and signal peptides. Protease assay and SDS-PAGE clearly showed that the N-terminal pro-domain plays an essential role in guiding the proper folding in trans of the enzymatically active conformation of aqualysin I. The N-terminal amino acid sequences of the purified enzymes were identical and had no signal peptides. These results indicate that independently expressed domains are secreted into the periplasmic space before the N-terminal pro-domain-assisted folding of the mature domain.