A rat hepatic cytosolic [3Hjmethyltrienolone (R1881) binding protein was studied under various conditions. This protein was also compared with the male-specific high capacity – low affinity estrogen-binding protein derived from the same cytosolic fraction. Analysis of the R1881 binding protein in adult (60–85 days old) male rat liver cytosol indicated the presence of a high affinity – low capacity binding site (Kd = 0.3 nM;Bmax = 5.9 fmol/mg) and a lower affinity – higher capacity component (Kd = 10.4 nM;Bmax = 131 fmol/mg). The latter component was eliminated by addition of triamcinolone or cortisol to the assay mixture. Steroid binding to the high affinity R1881 site was specific for testosterone, dihydrotestosterone, androstenedione, and mibolerone, with a moderate specificity to cyproterone acetate, flutamide hydroxide, and estradiol. Saturation studies indicated that these steroids were binding to the same or a similar high affinity component except for flutamide hydroxide which produced nonsaturable displacement. The high affinity site had no specificity for progesterone, diethylstilbestrol, or cortisol. Like the high capacity – low affinity protein, this protein was not present in the immature, adult, or 10-day ovariectomized adult female. However, unlike the high capacity – low affinity protein, it was present in low quantities in the immature male. In addition, castration of the adult for 18 h, 4 days, or 10 days or hypophysectomy for 10–17 days did not have a significant effect on the high affinity component compared with the controls. Testosterone administration to these animals did not alter this protein binding. These studies indicate that a specific, high affinity – low capacity androgen-binding protein exists in rat hepatic cytosol. Furthermore, this protein shows age and sex dependency, but its presence is not affected by altering gonadal or hypophyseal factors in the adult male.