Ronald L. Horst,* Peter M. Wovkulich, Enrico G. Baggiolini, Milan R. Uskokovic, George W. Engstrom, and Joseph L. Napoli abstract:(235')-l, 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 sepa-rated 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 (f]/2= 60 min). la, 25-Dihydroxy [3H] vitamin D3 26, 23-lactone appeared concurrently with the disappearance of (23S1)-1, 23, 25-trihydroxy [3H] vitamin D3. Experiments with radioinert compounds showed that 1, 25-dihydroxyvitamin D3 and (2351)-1, 23, 25-trihydroxyvitamin D3 were efficient pre-