A roundtable dialogue to discuss "NHANES Monitoring of Biomarkers of Folate and Vitamin B-12 Status" took place in July 2010. This article provides an overview of the meeting and this supplement issue. Although the focus of the roundtable dialogue was on the measurement of folate and vitamin B-12 status biomarkers in NHANES, this article also describes the relevance and importance of these issues for clinical and research laboratories. The roundtable identified the microbiological assay (MA) as the gold standard for measurement of serum and red blood cell folate concentrations. The roundtable noted that differences in results between the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA) that NHANES 1991-1994 and 1999-2006 used and the MA that NHANES 2007-2010 used will require adjustment equations to evaluate time trends. The roundtable found that the close agreement between the serum results for the MA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures supported the conversion to LC-MS/MS for serum folate in future NHANES. The roundtable recognized the uncertainty about whether subclinical vitamin B-12 deficiency is a public health concern but encouraged reinstatement of at least one circulating vitamin B-12 measure and one functional vitamin B-12 status measure in future NHANES. The use of serum vitamin B-12 and plasma methylmalonic acid would provide continuity with past NHANES. The roundtable supported the continued use of the National Institute of Standards and Technology (NIST) reference materials in NHANES biomarker analyses and the further development of additional reference materials by the NIST.