Abstract Despite the presence of known mutagens and carcinogens in cigarette smoke, there is currently no evidence to show that smoking, or exposure to cigarette smoke, can result in heritable genetic mutation. We show that male mice exposed to mainstream tobacco smoke (MTS) exhibit a significant increase in germ-line mutation frequency in spermatogonial stem cells. We exposed mature male mice to MTS for 6 or 12 weeks and investigated mutations arising in exposed spermatogonial stem cells at the expanded simple tandem repeat locus Ms6-hm. A generalized score test showed a significant treatment effect (P = 0.0214). Ms6-hm mutation frequency was 1.4 and 1.7 times higher in mice exposed to MTS for 6 and 12 weeks, respectively, compared with sham controls. The data suggest that mutations accumulate in the spermatogonial stem cells with extended exposures. Mutation spectra were identical between exposed and sham individuals, supporting the hypothesis that tandem repeat mutations arise through indirect mechanisms of mutation. Mutations in sperm that are passed on to offspring cause permanent, irreversible changes in genetic composition and can persist in future generations. Our research suggests that the consequences of smoking extend beyond the smoker to their nonsmoking descendents. [Cancer Res 2007;67(11):5103–4]