An examination of the pattern of lipid biosynthetic responses to isoniazid (INH) treatment of Mycobacterium tuberculosis and Mycobacterium smegmatis suggests that the mode of action of activated INH differs between these 2 organisms. Transformation of M. smegmatis with inhA on a plasmid construct conferred high-level resistance to INH, while the same construct failed to confer resistance upon M. tuberculosis. The inhA region from 2 clinical isolates whose resistance has been attributed to changes in the upstream promoter region has been cloned and was not sufficient to impart INH resistance to the level of the parent strain on sensitive M. tuberculosis. These putative mutant promoter elements appear to elevate expression levels of gene fusion reporter constructs, suggesting some noncausal connection between the observed mutations and the lipid metabolism of drug-resistant organisms. These results suggest that InhA is not the major target for activated INH in M. tuberculosis.