A radioimmunoassay for determination of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in serum, was compared with the radioreceptor assay using cytosol receptor from rachitic chick intestine in order to clarify whether differences in specificity can explain discrepancies in the clinical application of the two techniques. In the literature, seasonal fluctuations in serum 1,25(OH)2D3 are only observed when using radioimmunoassay. Treatment of anticonvulsant osteomalacia with vitamin D3 results in a marked increase in radioimmunologically measured 1,25(OH)2D3, but no effect on the serum 1,25(OH)2D level, measured by the radioreceptor assay, could be observed. The present study demonstrates that another unknown compound is coeluted on high pressure liquid chromatography with 1,25(OH)2D3, and recognised only by the antiserum. It can be concluded that the present radioimmunoassay cannot replace the cytosol receptor assay or either supplement by measuring the 1,25(OH)2D3 metabolite only.