Tracer specificity plays an important role in the radioimmunoassay (RIA) of steroid homones. In this paper, we describe the preparation and purification of125I-labeled cortisol derivative with a carboxymethyloxime-histamine bridge. The investigation on the method of purification showed that HPLC could be adopted for the routine preparation of a pure, homogeneous tracer. The retention time observed in HPLC for125I-histamine-CMO-cortisol conjugate could be used as an index for qualitative and quantitative assessments.