The gene coding for pyrazinamidase (PZase) activity, pncA, in Mycobacterium tuberculosis was recently cloned, and several mutations which correlate with in vitro resistance to pyrazinamide (PZA) have been identified. During the development of a clinical molecular assay for the detection of PZA resistance, two previously unreported mutations in isolates of PZA-resistant M. tuberculosis were identified. The assay that uses automated DNA sequencing is relatively rapid and allows the detection of any mutations present in the coding region of the pncA gene. The new mutations are both point mutations resulting in amino acid substitutions; 241T --> G results in F80V, and 511G --> C results in A171P. The identification of these mutations accentuates the utility of automated DNA sequencing in the clinical laboratory.