A new, rapid assay for la-hydroxylase has been developed using 2%hydroxy-[ 1a-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick la-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 'Hz0 can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 'Hz0 formed when 25-hydroxy-[l&'H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-'H]vitamin D3 substrate, production of labeled 1,25dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 'Hz0 produced from 25-hydroxy-[ la-'HIvitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for I ol-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations. a 1987 Academic Pres.s, Inc.