NNAL [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonol] is a valid biomarker of tobacco use. However; no study has assessed its use in distinguishing current smokeless tobacco (SLT) users from nonusers. Therefore, this study used aggregated data from the 2007-2008 and 2009-2010 waves of the National Health and Nutrition Examination Survey to determine an optimal threshold for identifying SLT users with NNAL. Optimal urinary total NNAL concentrations for discriminating SLT-only users from nonusers of any tobacco were determined using receiver operating characteristic analysis. Percentage agreement between self-reported SLT use status and NNAL levels was calculated overall and by sociodemographic characteristics. All analyses were weighted and performed with Stata, Version 11, and MedCalc for Windows, Version 9.5.0.0. In total, 264 individuals reported exclusively using SLT (and no other combustible tobacco product) within the past 5 days, whereas 14,824 were self-reported nonusers of any combustible or smokeless tobacco product. The optimal NNAL cutoff point was 34.0 pg/ml, which was associated with a high sensitivity (95.2%), specificity (93.4%), and overall correct classification rate (93.5%). The area under the curve was 98.3% and the corresponding Youden's Index was 88.7%. There was high agreement between the proposed NNAL cutoff point and self-reported SLT-only use (95.6%) and self-reported SLT nonuse (93.9%). The proposed cutoff point of 34.0 pg/ml had high sensitivity and specificity and may be used by clinicians and researchers to verify or detect recent SLT use. This study also indicated that self-reported SLT use among adults is a reliable measure and has high agreement with biochemical assessment.