It has been proposed that the antimitogenic action of progesterone (P₄) is mediated through a membrane receptor that has GABA_A receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P₄ with its gamma amino butyric acid_A (GABA_A) receptor-activating metabolite, 5α-pregnane-3α–21-diol-20-one (5α3α). These studies revealed that P₄ was more effective than 5α3α in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P₄ bound to SIGCs with an apparent dissociation constant (K_d) of 0.32 ± 0.09 μM, whereas 5α3α bound with an apparent K_d of 40 ± 19 μM. Further, the GABA_A antagonist, bicuculline, did not attenuate P₄'s antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known α chains of the GABA_A receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P₄ does not mediate its action via a GABA_A-like receptor. Additional studies revealed that P₄ regulated intracellular free calcium levels ([Ca² ]_i) as part of its antimitotic action. Specifically, P₄ maintained a basal [Ca² ]_i level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca² ]_i but also attenuated P₄'s antimitogenic effect. P₄'s actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P₄ (HP-P₄), which is cell impermeable, was as effective in blocking mitosis as P₄. Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P₄'s membrane-initiated actions.