A major vitamin D metabolite was isolated in pure form from blood plasma of chicks given either maintenance levels or large doses of vitamin D3. The isolation involved methanol-chloroform extraction and 5 column chromatographic procedures. The metabolite purification and elution position on these columns were followed by a competitive protein binding assay. The metabolite was identified using high- and low-resolution mass spectrometry, 270-MHz proton NMR spectrometry, UV absorption spectrophotometry, Fourier transform IR spectrophotometry and specific chemical reactions, as 3.beta.,25-dihydroxy-9,10-seco-5,7,10(19)-cholestatrieno-26,23-lactone. The trivial names 25-hydroxyvitamin D3 26,23-lactone or calcidiol 26,23-lactone were suggested for this compound.