25,26-Dihydroxyvitamin D3 [25,26(OH)2D3] is chemically synthesized as two stereoisomers: 25(R),26(OH)2D3 and 25(S),26(OH)2D3. Both the R and S configurations have been claimed to be the natural form. Previous studies, however, have not considered the possibility that the stereochemical configuration of the C-25 hydroxyl group could affect the binding of 25,26(OH)2D3 to the rat serum binding protein used in assays for the measurement of this metabolite. In our study, a 50% displacement of radiolabeled 25-hydroxyvitamin D3 ([3H]25OHD3) from its initial binding is achieved with 325 fmol/mL 25(R),26(OH)2D3 and 850-1000 fmol/mL 25(S),26(OH)2D3--a difference in potency of approximately 3-fold. The potency of the R isomer in displacing [3H]25OHD3 was similar to that of 25OHD3 or 24(R),25(OH)2D3. When 25,26(OH)2D levels were measured in 12 normal subjects with both the R isomer and the S isomer as standards, the results were 625 +/- 360 fmol/mL 25(R),26(OH)2D3 equiv and 1800 +/- 1130 fmol/mL 25(S),26(OH)2D3 equiv. To determine which stereoisomer is biosynthesized, two types of experiments were performed. In the first, radiolabeled 25,26(OH)2D3 was biosynthesized from [3H]25OHD3 by using chick renal mitochondria and compared to [3H]25OHD3 as a ligand in the rat serum binding protein assay, which was used to measure 25OHD3, 25(R),26(OH)2D3, and 25(S),26(OH)2D3. Initial binding of radiolabeled 25OHD3 and 25,26(OH)2D3 to the binding protein was comparable, as was their displacement by the nonradioactive metabolites, indicating that chick renal mitochondria produce primarily 25(R),26(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)