Introduction: The general secretory (Sec) pathway is the main pathway for protein transport. Sortase A has been shown to anchor surface protein containing an LPXTG motif to the cell wall. In order to know the distribution pattern of the Sec translocons and sortase A in Streptococcus mutans, we explored the subcellular localisation of SecA and sortase A. Methods: The anti-SecA antibody and anti-sortase A antibody were examined on Western blots to evaluate specificities. An indirect post-embedding immunogold method was used to determine the subcellular localisation of the Sec pathway and sortase A enzyme in S. mutans membrane. Results: Immunoblotting experiments showed that the anti-SecA antibody and anti-sortase A antibody specifically recognised a single band of about 95 kDa and 27 kDa, respectively in the S. mutans GS-5 cell lysates. Immunogold electron micrographs showed that small and large gold particles clustered together at a distinct microdomain. Conclusions: These data showed for the first time that Sec translocase and sortase A colocalised to a single locus in the cytoplasmic membrane of S. mutans GS-5. © 2007.