We describe a specific assay of 1,25-dihydroxy vitamin D3 in human serum, based on isotope dilution-mass fragmentography. We added [26-2H3]-1,25-dihydroxy vitamin D3 to a fixed amount of serum. The steroids were extracted with chloroform/methanol and purified by liquid chromatography. The purified materal was converted into the trimethylsilyl ether and analyzed. Unlabeled 1,25-dihydroxy vitamin D3 was quantitated from the ratio between the tracings at m/e 452 and 455. The two ions used correspond to loss of two trimethylsilyloxo functions from the molecular ion. Essentially the same results, with some problems of interference, were obtained when we used the more intense ions at m/e 131 and 134 (corresponding to cleavage between C-24 and C-25). The detection limit was about 5 ng/L of serum; the coefficient of variation was about 6%. The accuracy of the method was assessed by recovery experiments. 1,25-Dihydroxy vitamin D3 in sera from 15 healthy subjects was found to average 55 +/- 10 ng/L(+/- SD). We believe this represents the first determination of 1,25-dihydroxy vitamin D3 in serum by use of a method not based on radioimmunoassay or receptor assay. Slightly lower values have been reported by those latter techniques.