Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.