Arylamine N‐acetyltransferases (NATs; E.C 2.3.1.5) N‐acetylate arylhydralazine and arylamine substrates using acetyl coenzyme A. Human NAT2 acetylates and inactivates the antituberculosis drug, isoniazid (INH), and is polymorphic. We previously demonstrated that there is a homologue of human NAT2 in Mycobacterium tuberculosis, whose product N‐acetylates INH in vitro. We now demonstrate that the nat gene is expressed in M. tuberculosis and M. bovis Bacille Calmette‐Guerin (BCG), using reverse transcription–polymerase chain reaction and Western blotting. The NAT protein is active in M. bovis BCG in vivo, as detected by the presence of N‐acetyl INH in M. bovis BCG lysates grown in INH. Sequence analysis of the M. tuberculosis nat coding region reveals a single nucleotide polymorphism in 18% of a random cohort of M. tuberculosis clinical isolates, conferring a G to R change. The recombinant mutant protein appears less stable than the wild type, and has an apparent affinity for INH of 10‐fold less than the wild type. Modelling the change in M. tuberculosis NAT shows that the G to R change is close to the active site, and supports the experimental findings. Minimum inhibitory concentration data suggest that this polymorphism in nat is linked to low‐level changes in the INH susceptibility of M. tuberculosis clinical isolates.