There is increasing evidence that the corpus luteum has an important role in regulating its own demise. A series of experiments was performed to study the effects of luteal concentrations of progesterone on the functions of steroidogenic luteal cells. In the first experiment, steroidogenic small luteal cells (SLCs) were separated from endothelial cells, and it was determined that it was the SLCs that contained receptors for oxytocin. Treatment with progesterone (95 μM) for as little as 1 h decreased (P < 0.05) the percentage of SLCs responding to oxytocin (10 μM) with an increase in intracellular concentrations of calcium, and this effect continued for the duration of the experiment. In a second experiment, the response to oxytocin was increased (P < 0.05) by 3 h (but not 1 h) following progesterone removal, with a further increase by 16 h. The ability of 1 μM prostaglandin F_2alpha (PGF_2alpha) to increase intracellular concentrations of calcium was also decreased (P < 0.05) by progesterone treatment. By 3 h following removal of progesterone, the percentage of steroidogenic large luteal cells (LLCs) responding to PGF_2alpha was increased and not different from that observed in cells 16 h after progesterone removal. Finally, cyclodextrins (methyl-beta cyclodextrin [MbetaCD]) were used to remove cholesterol from the plasma membrane of luteal cells, and MbetaCD loaded with cholesterol was used to put cholesterol back into the plasma membrane of progesterone-treated cells. Treatment with MbetaCD reduced (P < 0.05) the responsiveness of SLCs to oxytocin and LLCs to PGF_2alpha. Use of cholesterol-loaded MbetaCD returned the responsiveness of both SLCs and LLCs treated with progesterone to that observed in vehicle (no progesterone)-treated controls. In summary, intraluteal concentrations of progesterone inhibit the ability of oxytocin to increase intracellular concentrations of calcium in SLCs and the ability of PGF_2alpha to increase intracellular concentrations of calcium in LLCs. The highest concentration of progesterone appears to act by influencing cholesterol content of the luteal cell membranes.