The ability of Mycobacterium tuberculosis to enter into a dormant state has resulted in one third of the world’s population being infected with latent tuberculosis. As such, the study of the molecular mechanisms that contribute to latency and reactivation is of utmost importance. M. tuberculosis encodes five resuscitation promoting factors, RpfA-E, which share homology with Micrococcus luteus Rpf, a muralytic enzyme implicated in reactivation of this organism from a dormant state. We have undertaken the combinatorial deletion of up to four of the five rpf genes from M. tuberculosis H37Rv to produce a set of deletion mutants that include three quadruple mutant strains that retain only rpfB, rpfD or rpfE. These strains were assessed for growth and survival in vitro and in macrophages and for virulence in mice. Our data indicated that although the quadruple mutants displayed normal growth and survival in liquid media and in human PBMCs, they showed reduced growth kinetics on solid media. Moreover, all three strains showed markedly reduced virulence in mice, with those carrying only rpfB or rpfD being unable to maintain chronic infection in vivo. These findings, together with preliminary data on the expression of the remaining rpf genes in the set of deletion mutant strains, suggest that the Rpf proteins of M. tuberculosis may have discrete functions.