The commonly accepted hypothesis explaining the mechanism of action of pyrazinamide (PZA) is based on the assumption that PZA-susceptible Mycobacterium tuberculosis strains produce pyrazinamidase, which hydrolyzes PZA to the antibacterial moiety pyrazinoic acid (POA). It is not clear whether POA has specific antimicrobial activity or the inhibition of growth caused by POA is due to its ability to lower the pH of the environment below the limits of tolerance of M. tuberculosis growth. We confirmed in this study that POA, depending on the concentration, lowered the pH of 7H12 broth (pH 6.0), which ranged from 5.8 at 120.0 micrograms/ml to 4.6 at 960.0 micrograms/ml. Therefore, we tested the inhibitory effects of different concentrations of POA in broth in which the final pH was adjusted to 5.6 by adding appropriate amounts of phosphoric acid or dipotassium phosphate. Under these conditions, we found a clear dose-response correlation, proving that POA does have specific antimicrobial activity. The MIC of POA at pH 5.6 was 240 to 480 micrograms/ml, 8- to 16-fold higher than the MIC of PZA under the same conditions and much higher than the concentrations achievable in humans. This suggests that the action of POA in an acid environment is a combined effect of its specific activity and its ability to lower the pH below the limits of tolerance of the target organism.