This study determined the effects of environmental tobacco smoke (ETS) on fetal growth by measuring neonatal birth outcomes and the extent of maternal DNA damage, and investigating the relationships among gene polymorphisms, genotoxicity, and pregnancy outcomes of expectant mothers who had exposed to tobacco smoke. This prospective study enrolled 685 pregnant women who completed an initial questionnaire at three central Taiwan hospitals between 2003 and 2004. Genotype analyses of CYP1A1, GSTT1, GSTM1, and NAT2 were performed from 421 women. A total of 398 women completed the follow-up analysis and successfully delivered a live single baby (n=384). Comet assay was performed for 18 smokers, 143 ETS-exposed subjects and 130 non-smokers to measure DNA damage. Analytical findings indicated that the levels of DNA damage among smokers and ETS-exposed subjects were significantly higher than that of non-smokers. DNA damage score in the ETS-exposed group was 84.3+/-44.3 and 63.5+/35.0 [corrected] for the nonsmoking group (p<0.001). Risk of DNA damage (DNA strand breakage, sister chromatid exchange, cell transformation and escalation of cytotoxicity) for subjects exposed to ETS was 7.49 times (adjusted odds ratio; 95% CI, 1.27-44.20) [corrected] greater than that of non-exposed to tobacco smoke at home. Average birth weight of neonates born to subjects with extremely serious DNA damage (within the 90th percentile, DNA damage score >or =129.5) was 141 g lighter than that of those with DNA damage score <129.5 (p=0.068) [corrected] The degree of DNA lesion was not related to metabolic polymorphic genes. The results of this study suggest that comet assay are reliable biomarkers for monitoring pregnant women exposed to tobacco smoke and indicate fetal growth effects from environmental exposure to tobacco smoke.