Measurement of serum 25-hydroxyvitamin D [25(OH)D] is generally considered to be a reliable indicator of vitamin D status. High variability in 25(OH)D measurements due to utilized test and assay technologies, non-equimolar detection of 25(OH)D2 and 25(OH)D3, interferences from other hydroxylated vitamin D metabolites, and the lack of a definite reference method often confounds proper assessment of vitamin D status (1, 2). Recently, two reference measurement procedures for 25(OH)D3 and 25(OH)D2 have been described using isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) (3, 4). The recent increase in diversity of 25(OH)D assays prompted us to evaluate the performance of chromatographic methods (two in-house ID-LC-MS/MS and HPLC (ClinRep, Recipe)), a protein binding method (Cobas-25(OH)D-total, Roche) and immunochemical methods (Liaison and RIA (Diasorin), iSYS (IDS), ADVIA Centaur (Siemens), and Architect i1000 and i2000 (Abbott)). Blood (serum-gel, S-Monovette, Sarstedt, Nümbrecht, Germany) was drawn at one site from random outpatients (N=60) after informed consent. Sample aliquots were prepared, frozen and transported to participating centers. Method comparison was performed according to CLSI-EP9 specifications. The Architect assays as well as the ADVIA Centaur assay evaluated in this study have been adjusted by the supplier relatively quickly after release on the market. All assays were compared to an established LC-MS/MS method (LC-MS/MS-a) (5). The accuracy of the second MS method (LC-MS/MS-b) was established by measuring the standard and a control with a reference measurement procedure (4). Details of the LC-MS/MS-b method have been published recently (6). The relative content of 3-epi-25(OH)D3 was measured by a modification of LC-MS/MS-a using a fluorophenyl column (7). Individual patient samples were measured in duplicate, with exception of HPLC, RIA and the Architect-i1000 where singular measurements were performed. For method comparison studies we applied Deming regression and Bland-Altman bias plots using EP Evaluator Release 9 (D.G. Rhoads Associates Inc., Kennett Square, PA, USA) or Analyse-it software program (Microsoft Corporation). Module CLSI EP9 Method Comparison was used for analysis of duplicate measurements, whereas module Alternate (Quantitative) Method Comparison was used for singular measurements.