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We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7alpha, 17alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecific interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7alpha- and 17alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with different types of spacer chains were used. The 3-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1-N-diamino-3, 6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3-oxo function. The 17alpha-position is accessible by the reaction of the 3-protected 4-androsten-17-epoxide with oxygen in the beta-position, followed by nucleophilic ring opening with cyanide which provides the 17alpha-cyanomethyl derivative. The key step is the regioselective ketal protection of the 3-oxo function of androst-4-ene-3,17-dione using a stannoxane catalyst. An alternative pathway for the insertion of biotin at the 19-position was established by the synthesis of 17beta-hydroxy-androst-4-en-3-one-19-yl carboxymethyl ether. After activation by the carbodiimide method, the compound reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1,6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7alpha-derivatives by use of omega-silyl protected hydroxylalkyl-modified Grignard reagents. A functional group interconversion using the Staudinger reaction transforms the azide function into a primary omega-amino group. The absolute configurations of the different biotinylated derivatives were investigated by (1)H NMR studies. For the 7alpha-biotinylated T series, additionally, an X-ray analysis proved the axial position of the spacer group. This results in a vertical orientation of the biotin moiety 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 7alpha-derivatives as optimal immunochemical tracers in competitive immunoassays. Biotinylated T derivatives should be also suitable for ligand-binding studies to the androgen receptor or to sex hormone-binding globulin.