A new metabolite of vitamin D was isolated from the blood plasma of chicks given large doses of vitamin D3. The isolation involved methanol-chloroform extraction and 4 column chromatographic steps. The metabolite was identified by high- and low-resolution mass spectroscopy, chemical derivatization and comigration with authentic standard as 3.beta.,24(R)-dihydroxy-9,10-seco-5,7,10(19)-cholestatriene [24(R)-hydroxyvitamin D3]. No detectable 24-(R)-hydroxyvitamin D3 was recovered from 16 l of plasma from chicks receiving physiologic levels of vitamin D3.