Estriol-6-(0-carboxymethyl) oxime (E 3-CMO) and estriol-4-azobenzoic acid (E 3-4-ABA) were linked to bovine serum albumin (BSA). Twelve rabbits were immunized, six with each E 3-BSA conjugate. All six E 3-6-CMO-BSA rabbits, but only one E 3-4-ABA-BSA animal, responded with useful antibody titers. All antisera exhibited good Ring D specificity. E 3-6-CMO-BSA (type 1) antisera cross-reacted up to 220 percent with 6-oxoestriol while the E 3-4-ABA-BSA (type 2) antiserum cross-reacted only 3.8 percent with this steroid. Neither type of antiserum cross-reacted with neutral steroids nor with estriol-16-glucosiduronate and estriol-3-sulfate-16-glucosiduronate, but both cross-reacted with estriol-3-sulfate and estriol-3-glucosiduronate. Both types of antisera could be utilized for a rapid and specific radioimmunoassay (RIA) of unconjugated E 3 in third trimester pregnancy plasma without need for further purification of the plasma extract. Blanks were negligible, sensitivity was sufficient, recovery was virtually complete by using 3H-E 3 as an internal standard, and precision was satisfactory. The measurements of unconjugated plasma E 3 concentrations in ninety apparently normal women between 29 and 40 weeks or gestation obtained by this RIA averaged 7.6, 10.2 and 16.7 ng/ml at 29 to 32, 33 to 36 and 37 to 40 weeks of gestation, respectively. The results obtained in this study indicate that antisera against E 3-6-CMO-BSA, despite their appreciable cross-reaction with 6-oxoestriol, are as useful for a rapid RIA of plasma unconjugated E 3 as antisera against E 3-4-ABA-BSA because very little, if any, 6-oxoestriol is present in late pregnancy plasma. As anti-E 3 titers were much higher and much more readily obtained in response to immunization with E 3-6-CMO-BSA than with E 3-4-ABA-BSA, E 3-6-CMO-BSA appears to be the preferable antigen to develop antisera for a rapid, yet specific, E 3 RIA.