Nonreplicating or dormant cells of Mycobacterium tuberculosis constitute a challenge to tuberculosis (TB) therapy because of their tolerance or phenotypic resistance to most drugs. Here, we propose a simple model for testing drugs against nongrowing cells that exploits the 18b strain of M. tuberculosis, a streptomycin (STR)-dependent mutant. Optimal conditions were established that allowed 18b cells to replicate in the presence of STR and to survive, but not multiply, following withdrawal of STR. In the presence of the antibiotic, M. tuberculosis 18b was susceptible to the currently approved TB drugs, isoniazid (INH) and rifampin (RIF), and to the experimental drugs TMC207, PA-824, meropenem (MER), benzothiazinone (BTZ), and moxifloxacin (MOXI). After STR depletion, the strain displayed greatly reduced susceptibility to the cell wall inhibitors INH and BTZ but showed increased susceptibility to RIF and PA-824, while MOXI and MER appeared equipotent under both conditions. The same potency ranking was found against nonreplicating M. tuberculosis 18b after in vivo treatment of chronically infected mice with five of these drugs. Despite the growth arrest, strain 18b retains significant metabolic activity in vitro, remaining positive in the resazurin reduction assay. Upon adaption to a 96-well format, this assay was shown to be suitable for high-throughput screening with strain 18b to find new inhibitors of dormant M. tuberculosis.Tuberculosis (TB) is one of the most important infectious diseases caused by a single pathogen and afflicts both healthy and immunocompromised individuals. One-third of the human population is reported to be latently infected with Mycobacterium tuberculosis, and millions of lives are lost every year (8). The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains (10), together with a deadly synergy with HIV, has worsened the global problem and forced the research community to look for new strategies to fight M. tuberculosis. As a consequence, several drug discovery programs have been established worldwide with the goal of finding new therapeutic agents that may complement or even replace the existing directly observed therapy short course (DOTS) for TB. These programs have to cope with the ability of the pathogen to enter the so-called dormant or latent phase (5, 6), characterized by phenotypic drug resistance (11). Complete control of TB will therefore require finding drugs that are effective against the reservoir of nongrowing tubercle bacilli and, hence, the necessity of modeling this state in vitro and in vivo in order to screen for active compounds.Latent TB is the result of complex host-pathogen interactions (26, 35), and mimicking this state may thus be partial and challenging. Different models have been proposed so far, each one based on a particular stress that the pathogen may encounter in the host. In particular, the oxygen depletion model mimics the hypoxic environment of the granuloma (30, 31) and the nutrient starvation model recapitulates the lack...