Within a few years, 25-hydroxyvitamin D (25-OHD) 1 has emerged as a high volume test in many regions. Several analytical issues have been recognized regarding this analyte, in particular variable co-detection of metabolites (25-hydroxyvitamin D 2; 3-epi-25-hydroxyvitamin D; 24,25-dihydroxyvitamin D), variable release of the analyte from its protein bonds and matrix effects in automated ligand binding tests, contributing to an often unsatisfactory correlation of high-throughput assays with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reference methods based on LC-MS/MS as well as reference materials have been introduced only very recently, achieving a truly significant improvement in the standardization of serum 25-OHD measurement. However, beyond these analytical issues in relation to 25-OHD, it should be scrutinized how biologically appropriate this inactive intermediate metabolite can actually describe vitamin D status as a surrogate marker -individually and at a population level. There is, for example, at present little knowledge regarding individual vitamin D requirement in relation to dietary calcium supply; the impact of genetic variation of vitamin D binding protein on serum concentrations; the impact of genetic variation in downstream metabolism and signaling of vitamin D on individual vitamin demand. Furthermore, there is no accepted approach to assess functional whole year vitamin D status, addressing the fundamental seasonal variation of endogenous generation of vitamin D in many regions. Consequently, additional functional markers should be considered when describing vitamin D status (parathyroid hormone, corrected serum calcium and phosphate, urinary calcium, well selected bone markers, etc.), with season-adapted sampling strategies. In conclusion, it should be recognized that there is substantial uncertainty in the currently used approach to characterize vitamin D status by singular measurement of 25-OHD using mainstream assays. It seems questionable to focus the worldwide debate on a widespread vitamin supplementation merely on cut-off results of this marker.