The activity of linezolid was studied against 55 Mycobacterium tuberculosis (42 susceptible, 3 isoniazid resistant and 10 isoniazid and rifampicin resistant), one Mycobacterium bovis and two multidrug-resistant M. bovis isolates using the standard 7H10 agar proportion and the ESP Culture System II methods. Both methods displayed similar MIC(90) values (minimum inhibitory concentrations for 90% of the organisms) of 0.5mg/L; however, the former method yielded slightly lower MIC(50) values (MICs for 50% of the organisms) (0.25mg/L) compared with the latter method (0.5mg/L). No differences were observed between susceptible and resistant isolates, including multidrug-resistant M. bovis isolates, with a MIC range of 0.12-0.5mg/L. The potential role of linezolid in tuberculosis patients requires further in vivo evaluation.