Rationale: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Objectives: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Methods: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Measurements and Main Results: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r s ¼ 20.77) and directly from sputum (r s ¼ 20.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r s ¼ 0.68) and semiquantitative colony counts (r s ¼ 20.56) was weaker than smear. Tests of paired same-patient sputum showed that highviscosity sputum samples contained 332 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Conclusions: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.Keywords: tuberculosis; molecular diagnostics; diagnostic techniques and procedures; diagnosis; clinical trial Measurements of bacterial load have long played an important role in tuberculosis diagnostics. Semiquantitative or quantitative measures of the number of Mycobacterium tuberculosis bacilli present within a clinical sample have been clinically useful for determining disease severity, assessing transmission risk, or monitoring therapy (1-3). Quantitative readouts have also aided in the investigation of potentially false-positive results (4-6).The Xpert MTB/RIF (Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin in less than 2 hours (7). The assay is contained in a small plastic cartridge, and it is run on the GeneXpert platform (Cepheid), a diagnostic The Xpert MTB/RIF assay is a rapid diagnostic test for tuberculosis and rifampin resistance. A number of studies have examined the sensitivity and specificity of the assay in various clinical settings and with various sample types. The test can also provide an estimate of the bacterial load present in the s...