The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosis with mutations in the katG gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosis are severe.