(23S)-1,23,25-Trihydroxyvitamin D3 was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyvitamin D3 by sequential chromatography through one Sephadex LH-20 column and three high-performance liquid chromatography systems. Ultraviolet absorption spectroscopy and mass spectrometry confirmed the structural assignment. One high-performance liquid chromatography system separated the R and S epimers of 1,23,25-trihydroxyvitamin D3 and indicated that the natural product had the S configuration. Plasma pharmacokinetic studies in rats showed that (23S)-1,23,25-trihydroxy[3H]vitamin D3 was rapidly cleared from plasma (t1/2 = 60 min). 1 alpha,25-Dihydroxy[3H]vitamin D3 26,23-lactone appeared concurrently with the disappearance of (23S)-1,23,25-trihydroxy[3H]vitamin D3. Experiments with radioinert compounds showed that 1,25-dihydroxyvitamin D3 and (23S)-1,23,25-trihydroxyvitamin D3 were efficient precursors to 1,25-dihydroxyvitamin D3 26,23-lactone both in intact and in nephrectomized rats. (25S)-1,25,26-Trihydroxyvitamin D3, however, was ineffective at raising plasma 1,25-dihydroxyvitamin D3 26,23-lactone concentrations. These results confirm the presence of extrarenal 1,25-dihydroxyvitamin D3 23(S)-hydroxylase(s) and demonstrate that C-23 hydroxylation of 1,25-dihydroxyvitamin D3 precedes C-26 hydroxylation in the formation of 1,25-dihydroxyvitamin D3 26,23-lactone. (23S)-1,23,25-Trihydroxyvitamin D3 had no intestinal calcium absorptive or bone calcium resorptive activity when dosed to vitamin D deficient rats at levels up to 500 ng. C-23 oxidation, therefore, appears to be a physiologic pathway of 1,25-(OH)2D3 metabolism and a major pathway for the deactivation of pharmacologic levels of 1,25-dihydroxyvitamin D3.