Background The measurement of 1α,25(OH) 2 D 3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere. Materials and methods During optimization of our in-house LC–MSMS method for serum 1α,25(OH) 2 D 3 a previously co-eluting isobaric interference was separated. The isobar was identified as 1β,25(OH) 2 D 3 by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D 3 analogs). 1β,25(OH) 2 D 3 showed specific cluster formation (water), not present in 1α,25(OH) 2 D 3 . 1β,25(OH) 2 D 3 was measured in serum of apparently healthy human volunteers (n = 20), patients with high serum 25-hydroxyvitamin D [25(OH)D] concentrations (>50 ng/mL) (n = 33 among which 4 with very high levels (>150 ng/mL)) and patients with kidney failure (n = 68; 39 stage 1–3, 29 stage 4–5). Pearson’s r was calculated for correlations and Mann-Whitney statistic to compare group medians. Results Median serum 1β,25(OH) 2 D 3 was 11 pg/mL in apparently healthy volunteers and increased to 20 pg/mL for serum 25(OH)D concentrations above 80 ng/mL (n = 22) (p < 0.0001). 1β,25(OH) 2 D 3 concentrations were significantly correlated to serum 25(OH)D concentrations (r = 0.85) for the combined results from healthy volunteers and patient sera (n = 53) (p < 0.0001). For patients with kidney failure, median serum 1β,25(OH) 2 D 3 was 7 pg/mL and not different from the median level in healthy volunteers (p = 0.06). The median concentration did not vary with different stages. Conclusions We present evidence for the widespread presence of 1β,25(OH) 2 D 3 , a new vitamin D metabolite, in human serum. The level increases with rising serum 25(OH)D concentrations and is particularly high in patients with very high 25(OH)D levels. We previously demonstrated that 1β,25(OH) 2 D 3 is a poor genomic agonist but a potent non-genomic antagonist of 1α,25(OH) 2 D 3. The clinical implications of the presence of this analog therefore require further exploration. [ABSTRACT FROM AUTHOR]