A soluble thermolabile protein with many characteristics of a progesterone receptor has been identified in ovaries of estrogen-stimulated, hypophysectomized, immature female rats. A potent synthetic progestin R5020 (17,21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione) and a progestin-receptor complex stabilizer (glycerol) were employed. After the incubation of [3H]R5020 with ovarian cytosol, fractionation on a Sephadex G-200 column revealed a peak of radioactivity which eluted with the void volume. This peak, which represented saturable binding, disappeared after heating (37 C for 20 min) and trypsinization. n the absence of glycerol, binding decreased by 84%. Scatchard analysis of the binding curve showed the R5020 binding to be of moderately high affinity (Kd = 14 nM), with 232 fmol binding sites/mg cytosol protein. Binding site number rose linearly with increasing cytosol protein concentration. The relative abilities of various steroids to inhibit [3H]R5020 binding were: R5020 > progesterone > estradiol > testosterone > cortisol > diethylstilbestrol. [3H]R5020 was not metabolized and did not bind specifically to serum. In summary, we have identified a protein with characteristics of a progesterone receptor in the cytoplasmic fraction of ovarian tissue.