The specialized RNA, tmRNA, is a central component of prokaryote trans-translation; a process that salvages stalled translational complexes. Evidence from other bacteria suggested that exposure to ribosome inhibitors elevated tmRNA levels, although it was unclear whether such changes resulted from increased tmRNA synthesis. Consequently, this study was initiated to determine the effect of ribosome inhibitors on the expression of tmRNA in mycobacteria. Exposure of Mycobacterium smegmatis to ribosome-targeting antimicrobial agents was associated with increased levels of the tmRNA precursor, pre-tmRNA, and mature tmRNA. For example, exposure to 16 μg mL−1 erythromycin for 3 h increased pre-tmRNA and tmRNA by 18- and 6-fold, respectively. Equivalent results were found following exposure of Mycobacterium bovis BCG to streptomycin. Exposure to antimicrobial agents with nonribosome targets did not affect tmRNA levels. The increased tmRNA levels were associated with increased output from the ssrA promoter, which controls tmRNA transcription, without evidence of a change in tmRNA degradation. These results suggest that the upregulation of tmRNA expression was an important response of bacteria to exposure to ribosome-inhibiting antimicrobial agents.