ABSTRACT Antibodies to estradiol were produced by immunization of sheep with 17β-estradiol-succinyl-BSA. The antiserum cross reacted readily with estrone and was used for radioimmunoassay of plasma estrone and estradiol. The method includes ether extraction, separation of estrone and estradiol by Sephadex LH-20 column chromatography and radio-immunoassay using a mixture of polymerized estradiol antibodies and tracer 3H-estradiol. Separation of the free and protein bound radio-activity was effected by centrifugation. The water blank of the method is 11 ± 1 picogram (pg). The lower limit of sensitivity is 50 pg. The coefficient of variation was 8–18% for estrone and 4–17% for estradiol. Analysis of plasma estrone and estradiol in 5 ovulatory menstrual cycles revealed a midcycle peak of estradiol in each subject.