Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.