The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to that of the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestral members of these families are found only within the ESAT-6 gene cluster regions. These ancestral members are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplications of the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated in virulence and are only found within the genomes of the pathogenic mycobacteria closely related to the M. tuberculosis complex. The aim of this study was to compare the sub-cellular localization of the later duplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that of the ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretion apparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genes from M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA as template.