We describe a multiple assay of the three main vitamin D metabolites, (250 \mathrm{HD}, 24,25(\mathrm{OH}){2} \mathrm{D}), and (1,25(\mathrm{OH}){2} \mathrm{D}), in (0.5 \mathrm{ml}) of serum, which does not require high-performance liquid chromatography. The assay involves extracting the serum with acetonitrile, separation and purification on a (\mathrm{C}-18 / \mathrm{OH}) cartridge and a Sep-Pak silica cartridge, and quantitation using (1,25(\mathrm{OH}){2} \mathrm{D}) receptors from calf mammary gland for (1,25(\mathrm{OH}){2} \mathrm{D}), and vitamin (D) binding protein for (25 \mathrm{OHD}) and (24,25(\mathrm{OH}){2} \mathrm{D}). For (25 \mathrm{OHD}, 24,25(\mathrm{OH}){2} \mathrm{D}), and (1,25(\mathrm{OH}){2} \mathrm{D}), the method is sensitive to (0.125 \mathrm{ng} /) tube, (0.025 \mathrm{ng} /) tube, and (0.5 \mathrm{pg} /) tube, with the (B{50}) occurring at (1 \mathrm{ng} /) tube, (0.2 \mathrm{ng} /) tube, and (8 \mathrm{pg} /) tube, respectively. The coefficients of variation (SD/mean (\times 100 %) ) intraassay ((n=8)) were (5.4,12.8), and (6.6 %) and interassay ((n=6) 12.3,10.8), and (8.6 %). The overall recovery was (80.4 \pm 5.5,58.0 \pm 6.3), and (77.4 \pm 5.6 %) (mean (\pm) SD, (n=40) ). The validity of the assay was confirmed by dilution test, analytical recovery of added vitamin D metabolites, and comparison with a standard assay using HPLC. This assay offers a simple, rapid, and precise method with which to determine the three main vitamin D metabolites in small serum samples, so it should be particularly useful in studies of pediatrics or small animals. 1994 Academic Press, Inc.