Dehydroepiandrosterone 3-sulfate (DHAS) binding to a cytoplasmic protein of rabbit uterine cervix was studied in vitro. When [3H]DHAS was incubated with the minced uterine cervix obtained at term pregnancy, it was bound specifically to the cytosol fraction. From Scatchard analysis, the binding capacity and dissociation constant were found to be 33 fmol/mg protein and 5.05×10-8M, respectively. Incubation of the prelabeled cytosol with isolated nuclei resulted in temperature-dependent transfer of DHAS to the nuclei. The cytoplasmic DHAS-binding protein after concentration with (NH4)2SO4 showed a sedimentation constant of 3.6-4.0S, while the DHAS-binding protein in the rabbit serum gave a value of 4.6S. The cytoplasmic DHAS-binding protein was eluted from Sephacryl S-300 mainly in the fractions corresponding to the molecular weight of 1.7×105, and in the void volume to a small extent in the presence of molybdate and glycerol. Dehydroepiandrosterone (DHA) inhibited approx. 50% of [3H]DHAS-specific binding, whereas 17β-estradoil 3-sulfate, estriol 3-sulfate, estrone 3-sulfate, 17β-estradiol, estriol, and 5α-dihydrotestosterone showed lower or no affinity for the DHAS-specific binding site. The content of the DHAS-binding protein in the cervix was found to increase with the progress of pregnancy.These observations suggest that rabbit uterine cervix contains a receptor-like DHAS-specific binding protein int he cytosol fraction which seems to be closely associated with the progress of pregnancy.