INTRODUCTION: It is perceived wisdom that within the host macrophage, Mycobacterium tuberculosis frequently encounters oxidative stress. Exposure of bacteria to reactive oxygen intermediates can have a mutagenic effect on the DNA. Various mutations are thought to arise as a consequence, including the oxidation of guanine residues, leading to G?C-->T?A substitution, and oxidation of cytosine resulting in a G?C-->A?T substitution. METHODS: We measured the relative contribution of oxidative stress by recording the percentage of single nucleotide substitutions reported in the genes rpoB and pncA that confer resistance to the antimicrobials rifampicin and pyrazinamide, respectively, and determined whether there is an excess of G?C-->T?A or G?C-->A?T substitutions. RESULTS: Out of 840 clinical isolates reported with single nucleotide mutations in the rpoB gene, 67% were G?C-->A?T changes, and 3% were G?C-->T?A substitutions. These figures were compared to the pncA gene, where out of 114 isolates, 30% of the single nucleotide mutations were G?C-->A?T transitions and 9% were G?C-->T?A changes. CONCLUSIONS: While there is an excess of G?C-->A?T changes in the rpoB gene, this was not the case in the pncA gene. Fifty-three percent of mutations within the rpoB gene were C-->T mutations of the type S531L. Although this mutation gives a fitness disadvantage, it is less than other common mutations, so it is more likely that that fitness is the determinant of surviving mutation rather than oxidative stress because of the small numbers of other C-->T and G-->A mutations at other sites (12%). There was no evidence of oxygen free radicals damaging the guanine bases in either gene.