In the United States, dietary supplements contribute a large proportion of micronutrient intakes. Therefore, it is important to collect accurate information on supplement use for studies of micronutrients and disease risk. This report describes the test-retest reliability and validity of a detailed, self-administered mailed questionnaire on vitamin and mineral supplement use. Participants (n = 220) completed the questionnaire at baseline and 3 months later. During an in-person interview, participants provided spot urine and blood samples, and interviewers transcribed nutrient information from their supplement bottle labels. The questionnaire had very good test-retest reliability for mean supplement intake over the past 10 years, with intraclass correlations ranging from 0.69 for beta-carotene to 0.87 for vitamin E. Pearson's correlation coefficients comparing current supplemental intakes from the questionnaire and interviews/label transcriptions were high, ranging from 0.58 for beta-carotene to 0.82 for chromium; however, for some nutrients, median intakes from the questionnaire were slightly lower than from the interviews. Beta-carotene, vitamin C, and vitamin E (alpha-tocopherol) showed clear linear trends of increasing blood concentrations with higher self-reported supplemental intakes (Pearson's correlation coefficients adjusted for potential confounding factors and diet = 0.31, 0.29, and 0.69, respectively; all p < 0.0001). Creatinine-adjusted spot urinary calcium values were not associated with supplemental calcium intakes (Pearson's r = -0.07). This self-administered questionnaire demonstrated high reproducibility and validity for collecting detailed information on supplement use.