We report here the first RIA for 1,25-dihydroxyvitamin D utilizing a radioiodinated (125I) tracer. This is also the first validated RIA for 1,25-dihydroxyvitamin D [1,25(OH)2D] that does not require sample prepurification by HPLC before the binding assay. The assay involves acetonitrile extraction, treatment of the crude extract supernate with sodium periodate, extraction and purification of endogenous 1,25(OH)2D by solid-phase chromatography, and finally, quantification by RIA. Calibrators were prepared in stripped human serum and processed exactly the same as samples, eliminating the need for internal control for procedural losses of endogenous 1,25(OH)2D. The assay consists of a 2-h room temperature incubation with the primary antibody, a 20-min incubation with a second antibody, and separation of bound from free by centrifugation. Assay results can be in hand with 5 h. The detection limit of the assay is 2.4 ng/L 1,25-dihydroxyvitamin D3. Results compare well with those from an accepted radioreceptor assay. Sample pretreatment with sodium periodate is absolutely essential before quantification by RIA; otherwise, concentrations of endogenous 1,25(OH)2D may be greatly overestimated.