Drug resistance is a growing problem in the treatment of mycobacterial disease. Resistance to isoniazid (INH), the most effective drug used to treat tuberculosis, has been associated with mutations in at least two genetic loci: the katG gene (which codes for catalase/peroxidase) and the inhA gene (which codes for an enoyl-reductase involved in mycolic acid synthesis). In this study, we use a novel and straightforward approach to inactivate the KatG enzyme in the cell and monitor its drug resistance phenotype. Exploiting the tetrameric nature of the enzyme, plasmids containing known katG mutants (defective in catalase/peroxidase activity) were introduced into wild-type (INH sensitive) Mycobacterium bovis (BCG). Expression of these mutant monomers in trans should inactivate the wild type enzyme by disrupting tetramer formation, or by forming inactive hetero-tetramers. Indeed, certain katG mutations do exhibit this trans dominant effect. Co-expression of these mutants with the wild type katG gene product results in reduction or elimination of enzyme activity (measured in cell extracts) with proportional increases in INH resistance. These trans dominant mutants show a direct correlation of INH resistance with mutations in the katG gene.