A hapten-radioimmunoassay for plasma oestradiol is described and information about the reliability of the method is given in detail. Oestradiol-3-hemisuccinate coupled to keyhole limpet haemocyanin is used for immunization of rabbits. The antiserum utilized for the assay is characterized by its titer, affinity and specificity. Following ether extraction and NaOH-light petroleum partition oestradiol is separated from crossreacting oestrogens by TLC. Oxidation of oestradiol on the plate is prevented by mercaptoethanol. To separate free and antibody bound ligand 250 μg dextran-coated charcoal per tube is used in the presence of bovine serum gammaglobulin (1 mg/ml). The between-assay precision based on 15 different determinations of control samples from normal adult male plasma was 9.4% (C. V.). The mean reagent blank value of 31 determinations was equivalent to 0.3 pg oestradiol and the detection limit in terms of the 99% confidence limit for a single blank value, was equivalent to 4.3 pg oestradiol. A procedure for detecting plasma blanks is described. Plasma oestradiol is separated from approximately all concomitant substances originally present in the sample by enzymatic conversion into oestrone and a second TLC. No plasma blanks could be detected with respect to normal adult male plasma. Normal values for adult males based on 51 subjects were characterized by a median of 17.2 pg/ml and the 95 percentiles of 9.5–27.6.