Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to .apprx. 90-, .apprx. 120-, and .apprx. 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (.apprx. 20% yield, 19% purity on the basis of one binding site per .apprx. 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-Pr fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not. Further evidence for the presence of the 90-kDa non-hormone-binding protein in the purified molybdate-stabilized nontransformed 8S-PR structure was demonstrated by (1) the presence of the three proteins in the 8.5S radioactive peak in a density gradient loaded with purified [3H]R5020-labeled PR, as in the 7.7-nm radioactive peak of this form analyzed by HPLC, (2) the shift of sedimentation coefficient of the purified 8S-PR in density gradient analysis, occurring after incubation with a specific antibody against a non-hormone-binding, calf uterine 90-kDa protein, and (3) the presence in this shifted peak of both 90-kDa protein and 120- and 85-kDa receptor proteins. In the course of this work, it was verified that 0.3 MK Cl added in cytosol in the absence of molybdate dissociated the 8S-PR complex, and purified 120- and 85-kDa progestin binding proteins were obtained by hormone-specific affinity chromatography of the salt-treated cytosol. In addition, despite the stabilization effect of molybdate ions, partial dissociation occurred during chromatography, and "4S-PR" was found in the flow-through of the DEAE-Sephacel column; after further purification by DNA-cellulose, it yielded the 120- and 85-kDa progestin binding units with 60% purity. In summary, the results demonstrated that, as for the nontranformed avian 8S-PR [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023], the nontransformed 8S from of the rabbit uterus PR includes a non-hormone-binding 90-kDa protein.