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Binding ability of androgen-binding protein (ABP) in concentrated media from Sertoli cell cultures and in various preparations of cytosols from rat testicular tissue have been estimated using dialysis and polyacrylamide gel electrophoresis (PAGE). Equilibrium dialysis in the presence of 1 nM-17β-hydroxy-5-androstan-3-one appears to be the most convenient and reliable method for estimation of ABP if no other binding proteins are present. PAGE can be applied for estimation of ABP in samples which also contain other steroid-binding proteins. Estimated binding activities in culture media measured by PAGE and dialysis were essentially similar. Binding of androgens to ABP estimated with PAGE in cytosols prepared after sonication of testicular tissue was much lower than in cytosols obtained after homogenization using a Potter–Elvehjem homogenizer. When ABP was added to cytosols prepared after sonication or homogenization, approximately 50 and 90% respectively of the original binding of androgens to ABP was recovered.