The preparation and characterization of an immunogen of estradiol is described. The 17-monosuccinate of estradiol (E-17MS) was coupled to bovine serum albumin (BSA) using carbodiimide. The immunogen was characterized for its chemical structure by various analytical methods. Rabbits were immunized, and the antisera collected was checked for immunoreactivity. A novel radiotracer was prepared by conjugation of E-17MS to tyrosine methyl ester hydrochloride (TME) using carbodiimide and radiolabeled by sodium iodide125. Incubation of raw antiserum was carried out in the presence of E-17MS-TME125I. Separation of free tracer from the bound form was achieved by a dextran-coated charcoal suspension. The percentage binding obtained was around 34%, thereby confirming the immunogenic potential of the antigen prepared.