Context: Recognition that vitamin D might be associated with many chronic diseases has led to large-scale epidemiological and clinical studies. Dried blood spots (DBS) are a useful resource for these studies. Consequently, accurate, efficient, and inexpensive assays to quantify 25-hydroxyvitamin D (25OHD) in DBS are required. Copyright Objective: This study evaluated the validityandreliability of a liquid chromatography-tandem mass spectrometry assay for measuring 25OHD in archived DBS and compared measurements of 25OHD in DBS with those in plasma. Design and Participants: Sixty-two participants in the Melbourne Collaborative Cohort Study who had plasma and matching DBS stored since study entry in the early 1990s were randomly selected for a study calibrating 25OHD concentrations in DBS with plasma. As part of a study of vitamin D and mortality, cancer, and diabetes, we also assessed the reliability of measurements from DBS using 500 replicates placed randomly within 31 batches run over 15 months. Outcome Measure: 25OHD concentrations were measured by liquid chromatography-tandem mass spectrometry. Results: There was good agreement between measurements of 25OHD from DBS and plasma; R2= 0.73 from a regression of plasma concentration on DBS concentration. The within-batch and between- batch intraclass correlations from the 500 replicate measurements were 0.82 (95 confidence interval, 0.80, 0.85) and 0.73 (95 confidence interval, 0.68, 0.78), respectively. Conclusions: Measuring 25OHD in DBS is a valid and reliable alternative to measuring 25OHD in sera or plasma. A simple calibration model was developed to convert measurements from DBS to equivalent plasma measurements, thus enabling comparisons against clinical reference ranges and with studies using sera or plasma samples.