A 4.0 kb EcoRI fragment of Bacillus subtilis conferring thiostrepton resistance was cloned and characterized. By nucleotide sequencing of the relevant region, six open reading frames were established, which corresponded to a part of spo0H, a ribosomal protein gene (rpmG), an unidentified open reading frame (orfE), a transcription antiterminator gene nusG, and ribosomal protein genes rplK and rplA. The orfE-encoded 59-amino-acid polypeptide had a low, but significant, sequence similarity with the carboxy-terminal region of the Escherichia coli SecE protein. A cold-sensitive secE mutation of E. coli was complemented by the plasmid-borne orfE sequence. Furthermore, the normal processing of a proOmpA protein was observed when the secE cold-sensitive strain carried an orfE plasmid, indicating that orfE is the secE homologue of B. subtilis. The B. subtilis secE has only one transmembrane sequence compared to the three in E. coli.