Rationale: Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. While mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesised that tuberculous sputum might include a population of Rpf-dependent Mtb cells. Objectives: To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. Methods: In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay to that determined by limiting dilution, Most-Probable Number (MPN) assay in the presence or absence of Rpf preparations. Measurements and Main Results: In 20 of 25 pre-chemotherapy samples from separate patients, 80-99.99% of the cells demonstrated by cultivation could only be detected with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf-dependency was lost following primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. Conclusions: Smear-positive sputum samples are dominated by a population of Mtb cells that can only be grown in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.