The metabolites of the tobacco-specific nitrosamine NNK form DNA adducts in animal models. A previous report indicates that NNK could cause damage to the mitochondrial as well as nuclear genome in rats (Stepanov and Hecht, 2009 Chem. Res. Toxicol. 22: 406). Using a different DNA damage detection technology, we tested whether this could be repeated in the nematode Caenorhabditis elegans; we also evaluated whether mitochondrial function would be affected. We treated N2 strain (wild-type) nematodes with NNK in liquid culture. Quantitative PCR was applied to analyze NNK-induced nuclear and mitochondria DNA damage. This assay has the advantage of measuring all DNA lesions that inhibit the DNA polymerase, and normalizes results to mitochondrial DNA copy number. Our results confirm that NNK causes both nuclear and mitochondrial DNA damage, but surprisingly nuclear DNA damage was greater than mitochondrial DNA damage in C. elegans. To test whether the mitochondrial DNA damage was associated with mitochondrial dysfunction, we used a transgenic nematode strain that permits in vivo measurement of ATP levels and found lower levels of ATP in NNK-exposed animals when compared to the unexposed controls. To test whether the lower levels of ATP were due to the inhibition of respiratory chain components we investigated oxygen consumption in whole C. elegans and found reduced oxygen consumption in exposed animals when compared to the unexposed controls. Our data suggest a model in which NNK causes damage to both C. elegans nuclear and mitochondrial genomes, and support the hypothesis that the mitochondrial damage is functionally important. These results also represent a first step in developing this genetically tractable organism as a model for assessing NNK toxicity.