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BACKGROUND: Acrolein is a highly reactive α,β unsaturated aldehyde and respiratory irritant. Acrolein is formed during combustion (e.g., burning tobacco or biomass), during high-temperature cooking of foods, and in vivo as a product of oxidative stress and polyamine metabolism. No biomonitoring reference data have been reported to characterize acrolein exposure for the U.S. population. OBJECTIVES: Our goals were to a) evaluate two acrolein metabolites in urine--N-acetyl-S-(3-hydroxypropyl)-L-cysteine (3HPMA) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (CEMA)--as biomarkers of exposure to acrolein for the U.S. population by age, sex, race, and smoking status; and b) assess tobacco smoke as a predictor of acrolein exposure. METHODS: We analyzed urine from National Health and Nutrition Examination Survey (NHANES 2005-2006) participants [greater than or equal to]12 years old (n = 2,866) for 3HPMA and CEMA using ultra-high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/ESI-MSMS). Sample-weighted linear regression models stratified for nontobacco users versus tobacco smokers (as defined by serum cotinine and self-report) characterized the association of urinary 3HPMA and CEMA with tobacco smoke exposure, adjusting for urinary creatinine, sex, age, and race/ethnicity. RESULTS: 3HPMA and CEMA levels were higher among tobacco smokers (cigarettes, cigars, and pipe users) than among non-tobacco users. The median 3HPMA levels for tobacco smokers and non-tobacco users were 1,089 and 219 µg/g creatinine, respectively. Similarly, median CEMA levels were 203 µg/g creatinine for tobacco smokers and 78.8 µg/g creatinine for non-tobacco users. Regression analysis showed that serum cotinine was a significant positive predictor (p < 0.0001) of both 3HPMA and CEMA among tobacco smokers. CONCLUSIONS: Tobacco smoke was a significant predictor of acrolein exposure in the U.S. population. Environ Health Perspect 123:1302-1308; http://dx.doi.org/10.1289/ehp.1409251, Introduction Acrolein is a chemical contaminant that is ubiquitous in the environment. It is formed from carbohydrates, vegetable oils, animal fats, and amino acids during heating of foods and by [...]