Mutations within the pncA gene can cause pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. To study further the mutational events leading to PZA resistance, a H37Rv PZA-susceptible strain was used to derive PZA-resistant mutants in vitro. This parental strain was grown at gradually increased concentrations of PZA (from 200 up to 500 microg/ml). A total of eight variant strains were selected for further study. PZA resistance was selected at 250 microg/ml or higher. Seven of them lost PZase activity and hence were PZA resistant. The remaining strain has shown a functional PZase and hence was sensitive to PZA after being exposed to 200 microg/ml PZA. Sequence analysis shows different novel mutations within the pncA gene of these variants. Two silent mutations at nucleotide 6 (G --> A) and 300 (C --> T) were found in the PZA-sensitive variant. Among the seven PZA-resistant variants, there were three deletion mutations found at nucleotide 78 (G), 79 (A), and 143 (A), all resulting in a frameshift, and one nonsense mutation at nucleotide 102 (C --> G), creating a stop codon TAG. It was observed that the mutations found in the pncA gene of these PZA-resistant mutants show a distinctive pattern: mutation positions moved toward the beginning of the open reading frame correlating with the increase in PZA concentration, suggesting a possible relationship between the increase of PZA concentration and the early occurrence of mutations on the pncA gene.