Progesterone (P₄) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P₄ receptor. It has been proposed that P₄ mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P₄ binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P₄ binding site of the nuclear P₄ receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P₄, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P₄ in the presence or absence of nonlabeled P₄. This study demonstrated that the 60-kDa protein specifically binds P₄. Scatchard plot analysis revealed that ³H-P₄ binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a K_d of 360 nM. ³H-P₄ binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P₄ inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P₄ binding protein functions as a low-affinity, high-capacity membrane receptor for P₄.