The four stereoisomers of 25-hydroxyvitamin D(3) 26,23-lactone were synthesized by a stereoselective lactonization method. Natural 25-hydroxyvitamin D(3) 26,23-lactone was produced from 25-hydroxy-[3alpha-(3)H]vitamin D(3) by in vitro incubation of kidney homogenate prepared from vitamin D-supplemented chickens or was isolated from the serum of rats given 1,25-dihydroxyvitamin D(3) and 25-hydroxy-[3alpha-(3)H]vitamin D(3). The four synthetic isomers and the naturally produced 25-hydroxyvitamin D(3) 26,23-lactone were subjected to high-performance liquid chromatography in a system capable of separating the four isomers. The natural lactone comigrated with synthetic (23S,25R)-25-hydroxyvitamin D(3) 26,23-lactone, establishing it as the natural vitamin D(3) metabolite.