Antisera for radioimmunoassay of 17alpha-ethynyl estradiol and mestr anol were prepared and tested. The antisera were prepared by immunizing rabbits with 6(O-carboxymethyl) oxime-bovine serum conjugates from 6-oxo-17alpha-ethnyl estradiol and 6-oxomestranol. Antisera raised to 17alpha-ethynyl estradiol by employing steroid-protein conjugates coupled at either carbon-6 or -7 proved to be highly specific and showed little cross-reaction with mestranol. Antisera for mestranol also showed little cross-reactivity while the antibody to the 6-linked agent showed only moderate cross-reaction to 6-hydroxymestranol but an avidity for the 6-oxomestranol which exceeded that of the reference compound. This assay has been employed in measuring these substances after ingestion of oral contraceptives.