A few studies have reported on the measurement of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in bone, using chloroform/methanol extraction and radioreceptor assay. As the significance of bone 1,25(OH)2D3 content was not defined in any of these reports, the objective of the current investigation was to determine whether 1,25(OH)2D3 may be stored in skeletal matrix. Bone powder samples from the iliac crest were extracted in ethylacetate/cyclohexane and 1,25(OH)2D3 isolated from the extract by means of Sephadex LH-20 and high pressure liquid chromatographic separation and subsequently measured by radioimmunoassay (RIA). Within the detection range of the RIA, no 1,25(OH)2D3 could be measured, suggesting that 1,25(OH)2D3 is not stored in skeletal matrix. Vitamin D bone concentrations previously measured may therefore have reflected plasma contamination. Consistent with this hypothesis, only traces of skeletal 1,25(OH)2D3 binding protein were measured when compared with serum values. Although 1,25(OH)2D3 may act as a potential local determinant of bone remodeling, there is no evidence supporting a delayed paracrine function by matrix-derived 1,25(OH)2D3.