Tuberculosis is responsible for one in four of all avoidable adult deaths in developing countries. Increased frequency and accelerated fatality of the disease among individuals infected with human immunodeficiency virus has raised worldwide concern that control programmes may be inadequate, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in several recent fatal outbreaks in the United States. Isonicotinic acid hydrazide (isoniazid, INH) forms the core of antituberculosis regimens; however, clinical isolates that are resistant to INH show reduced catalase activity and a relative lack of virulence in guinea-pigs. Here we use mycobacterial genetics to study the molecular basis of INH resistance. A single M. tuberculosis gene, katG, encoding both catalase and peroxidase, restored sensitivity to INH in a resistant mutant of Mycobacterium smegmatis, and conferred INH susceptibility in some strains of Escherichia coli. Deletion of katG from the chromosome was associated with INH resistance in two patient isolates of M. tuberculosis.