Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5" ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.