Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence and the infection of host cells by mycobacteria. In the studies presented here, we have expressed the PE_PGRS 33 gene in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that it survives better in macrophage cultures, in vitro as well as in mice after intraperitoneal administration, than the parental strain containing the vector only or a strain expressing only the PE domain of PE_PGRS 33. In macrophages, enhanced colonization by the M. smegmatis expressing PE_PGRS 33 was associated with macrophage aggregation and clearance of macrophage monolayers, visible cell necrosis and significantly greater levels of TNF (TNF-alpha) in the cultures compared with controls. The presence of macrophage cell necrosis was confirmed by measurement of significantly greater levels of lactate dehydrogenase and nucleosomes in the supernatants of the macrophage cultures infected with M. smegmatis expressing PE_PGRS 33. Antibodies directed against TNF partially reduced cytolysis, suggesting that this cytokine is critical but not sufficient for the observed macrophage necrosis and enhanced mycobacterial survival. These results extend earlier observations, which suggested that PE_PGRS proteins may have a role in the pathogenesis of mycobacterial disease and that there may be a specific role for these proteins in influencing host cell responses to infection.