OBJECTIVE: Societal pressures against smoking during pregnancy may lead to a reduction in disclosure of smoking status. The objective of this study was to compare prevalence of smoking at prenatal intake by self-report with anonymous biochemical validation. METHODS: Women receiving care at the Duke Obstetrics Clinic from February 2005 through January 2006 were eligible for evaluation. Self-reported smoking and urine samples were obtained anonymously at prenatal intake. The NicCheck™ I semi-quantitative dipstick was used to detect urinary nicotine, cotinine, and 3-hydroxycotinine. The difference, with 95% confidence interval, between the proportions of smokers by self-report and urine testing was calculated for (1) high-positive vs. low-positive and negative results combined and (2) any positive vs. negative results. RESULTS: Among 297 subjects, self-reported smoking was 18.2 vs. 14.8% for low-positive and negative results combined with an absolute difference of 3.4%, [-2.9%, 9.6%]. When comparing self-report with any positive result (43.1%), the absolute difference was 24.9%, [17.4%, 32.1%]. CONCLUSIONS: Our findings suggest that most pregnant women disclose their smoking and many nonsmokers may have significant second-hand exposure. Universal urinary cotinine screening of pregnant women could aid in appropriately counseling women about second-hand exposure as well as monitoring women at high risk for adverse pregnancy outcomes.