The vitamin K external quality assurance scheme (KEQAS) aims to assist in the harmonization of phylloquinone (vitamin K(1)) analysis in order to improve the comparability of clinical and nutritional studies. Serum samples were despatched to 17 groups from eight countries during 2000-2006. Using pilot data (1996-1999), an analytical performance target of 20% absolute difference from the all-laboratory trimmed mean (ALTM) was assigned and formed the basis for interlaboratory comparison. Assay specificity, analytical bias and assay performance were evaluated. From 21 batches of samples distributed, 414 results were reported of which 2.7% were outliers. The mean interlaboratory absolute difference from the ALTM was 21.7% with 47% of groups consistently meeting the performance target. The mean interlaboratory coefficient of variation was 29.6%. The false positive rate for phylloquinone depleted samples was high at 35%. Bias was found to be independent of HPLC-detector type (fluorescence vs electrochemical). Assay characteristics for the measurement of phylloquinone in human serum compare favourably with methods for analytes at equivalent concentrations. The high proportion of false positive results suggest that poor assay specificity at low phylloquinone concentrations is a common problem, which in the clinical setting could lead to underreporting of vitamin K deficiency.