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Biotinylated 17beta-estradiol (E2) derivatives are helpful probes for a better understanding of biospecific E2 interactions with steroid-binding proteins such as the estrogen receptor and anti-steroid antibodies. We describe synthetic strategies for the biotinylation of E2 toward the 3, 6alpha, and 7alpha positions using biotinyl-N-hydroxysuccinimide esters with different spacers, varying in structure and chain length. Key reaction for biotinylation at the 3 position is the regioselective ether formation of the phenolate E2 anion with a linker mesylate without protecting the 17beta-hydroxyl group. The 6alpha position is accessible via a 3,17beta protected 6alpha-hydroxy E2, prepared by stereospecific sodium borohydride reduction of 6-oxo E2. Direct cyanoethylation of the alcohol followed by reduction to the amine allows the biotinylation to 6alpha-O-coupled cyanoethyloxy linker E2 derivatives. Alternatively, 6alpha-O-coupled cyanoalkyloxy polyether linker E2 probes are obtained by a Williamson ether synthesis of the alcohol precursor with omega-t-butyl-dimethylsilyloxy-5-oxa-nonylmesylate. Cyanoethylation of the desilylated compound and further reduction of the nitrile led to the terminal amine. Reductive amination of the 3, 17beta acetylated 6-oxo E2 compound with 6-cyanoethyloxyhexyl ammonium acetate yields in a mixture of 6alpha/beta-N-alkylated E2 nitriles. The epimers are separated by reversed-phase HPLC and the 6alpha-compound subsequently reduced to the terminal amine. The 7alpha-biotinylated E2 compound is derived from 7alpha-(11'-undecyl-N-methyl-N-butylamide) E2, which is already known from literature. Subsequently, the 3 and 17beta positions are protected, and the amide is reduced to the 7alpha-(11'-undecanol) compound. Further cyanoethylation and reduction led to the 11'-amino-ethyloxyundecyl E2. Using (1)H NMR analysis, it could be shown that the biotin moiety of the biotinylated 6alpha- and 7alpha-E2 derivatives has an axial position which results in a vertical orientation of the substituent toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 6alpha- and 7alpha-derivatives as optimal tracers in competitive immunoassays.