23,25-Dihydroxyvitamin D3 was isolated from vitamin D-toxic pig plasma by sequential chromatography through two gravity columns and three high performance liquid chromatography systems. Two of the high performance liquid chromatography systems separated the R and S diastereomers of 23,25-dihydroxyvitamin D3 and demonstrated that the metabolite has the 23S configuration. Ultraviolet absorbance and mass spectroscopy of the pure metabolite and mass spectroscopy of its trisilylated derivative confirmed the structural assignment. 23S,25-Dihydroxyvitamin D3, 23R,25-dihydroxyvitamin D3, 25S,26-dihydroxyvitamin D3, and 25R,26-dihydroxyvitamin D3 were assessed for their ability to produce 25-hydroxyvitamin D3-26,23-lactone in vitamin D2-toxic rats. On 23S,25-dihydroxyvitamin D3, of the naturally occurring compounds, was able to increase the plasma lactone concentration. This metabolite was a more efficient precursor than 25-hydroxyvitamin D3, suggesting that 23S-hydroxylation is a rate-limiting step in 25-hydroxyvitamin D3-26,23-lactone formation. 23S,25-Dihydroxyvitamin D3 was not detected in 25-hydroxyvitamin D3-dosed rats, indicating that the former is rapidly metabolized. Nephrectomized rats had a diminished but significant ability to synthesize 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3. Nephrectomy did not affect synthesis of 25-hydroxyvitamin D3-26,23-lactone from 23S,25-dihydroxyvitamin D3. These results demonstrate that vitamin D3 23S-hydroxylase(s) are also located extrarenally and that extrarenal tissues are quantitatively important to 25-hydroxyvitamin D3-26,23-lactone synthesis.