Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmokers. Among the individuals sampled were three pairs of monozygotic twins in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years. Individual point mutations present at frequencies as low as 10(-6) were detected. Partially denaturing electrophoresis was used to separate mutant from nonmutant sequences on the basis of their melting temperatures, and the target sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch intermediates or DNA adducts present in the cellular DNA were converted to mutants during PCR. Hot spot mutations were clearly observed in bronchial epithelial cells, and the same hot spots were observed consistently in different samples. Significant numerical variability in the mutant fractions for individual mutants was observed among samples and are ascribed to unequal mitochondrial segregation in stem and transition cells. The mutational spectra in smokers' samples did not differ significantly from the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA. No smoking-specific hot spots were detected. The overall mutant fractions in smokers' samples were not elevated compared to those of nonsmokers. As much variability was observed between two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker. These findings demonstrate that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial epithelial cells. Endogenous factors (e.g., DNA replication errors or DNA damage by endogenous reactive chemicals) are suggested to be more likely to represent the most important contributors to mitochondrial mutagenesis.