question: Vitamin D(3) is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH)(2)D(3), 25-hydroxyvitamin D(3) (25(OH)D(3)), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and <0.05 as a false discovery rate, in this ex vivo approach the maximal physiological concentration of 25(OH)D(3) (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D(3) regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH)(2)D(3). The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D(3). In conclusion, 100- and 1000-fold higher 25(OH)D(3) concentrations than the reference 10 nM 1,25(OH)(2)D(3) are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH)(2)D(3).