To perform a multicentre study evaluating the performance of the nitrate reductase assay (NRA) using liquid medium for the detection of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, amikacin, kanamycin and capreomycin. The study was carried out in three phases. Phase I determined the MIC of each drug. Phase II established the critical concentration of each drug. Phase III validated critical concentrations for the six drugs tested by the NRA using liquid medium compared with the agar proportion method or MGIT 960 system at each site. The critical concentrations for the six drugs used in the NRA are as follows: rifampicin, 1 mg/L; isoniazid, 0.2 mg/L; ofloxacin, 2 mg/L; amikacin, 2 mg/L; kanamycin, 5 mg/L; capreomycin, 2.5 mg/L. Phase III: Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 97%–100% for rifampicin, 96.8%–99.2% for isoniazid, 98%–100% for ofloxacin, 96.8%–98.5% for amikacin, 96.4%–99.5% for kanamycin and 96.8%–100% for capreomycin. Results for NRA using liquid medium were obtained in a median time of 7 days. NRA performed in liquid medium offers a rapid, economical and feasible method for detection of M. tuberculosis resistance to first- and second-line drugs in resource-limited settings.