Mutations within the pncA gene coding for pyrazinamidase of Mycobacterium tuberculosis can cause pyrazinamide (PZA) resistance. The effect of drug concentrations on PZA resistance in a clinical isolate of M. tuberculosis was studied in vitro. Serial passage at gradually increased concentrations of PZA from 200 to 500 microg/ml was performed using BACTEC radiometric method. Thirteen in vitro-selected variant strains were assembled and sequence analysis showed that 12 of the 13 variants had a novel single point mutation within the pncA gene by deletion at nucleotide 381 (G), codon 127. This lead to a frameshift that affected the function of the pyrazinamidase resulting in PZA resistance regardless of different PZA concentrations used. One variant had a silent mutation at nucleotide 6 (G-->A) and remains PZA sensitive. We conclude that the mutation location found is an important position for full resistance, at least in this strain. The lack of further mutations even after exposure to higher PZA concentrations implies a critical value for development of resistance-a level exceeded in tissues in clinical treatment regimes.