Specific binding of [3H]5α-dihydrotestosterone (DHT) and [3H]estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm*y♂), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4–5 S in gradients containing 0.5M KCl. KD's (mean ± SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y ♂ are 1.1 ± 0.4, 0.9 ± 0.4, and 0.8 ± 0.1 × 10-9M, respectively. DHT binding activity in brain cytosol from tfm/y ♂ mice is reduced to about 20–30% of that from their normal littermates, as is the case for tfm/y ♂ kidney cytosol. The residual androgen receptor in tfm/y ♂ brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y ♂ mice. KD's (mean ² SE) for estradiol binding are 1.6 ± 0.5 × 10-10M for castrated males, 2.4 ² 0.4 × 10-10M for females, and 1.8 ² 0.4 × 10-10M for tfm/y ♂. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the K1 is 8–9 × 10-9M. The decrease in the number of DHT binding sites in the brain of tfm/y ♂ mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol. (Endocrinology98: 864, 1976)