Abstract The effects of prostaglandin F 2α (PGF 2α administration on the utilization of low density lipoprotein (LDL) and progesterone secretion were examined in dispersed luteal cells from rat ovaries. Immature rats were rendered pseudopregnant with administration of pregnant mare serum gonadotropin and human chorionic gonadotropin. Animals were sacrificed at different times after PGF 2α (5 mg/kg) or vehicle administration on day-5 of pseudopregnancy. Administration of PGF 2α in vivo decreased human chorionic gonadotropin (hCG) binding to luteal cell membranes in vitro but enhanced binding of LDL. Utilization of labelled cholesterol for steroid synthesis from reconstituted LDL [( 3H)-CL-LDL] by dispersed luteal cells was enhanced following PGF 2α administration. This suggests that the LDL pathway is not suppressed during prostaglandin induced luteolysis. Progesterone and total progestin secretion in response to N 6-2′-0-Dibutyryladenosine 3′5′-cyclic monophosphate (cAMP) was decreased at 2,4 and 24 hours following PGF 2α administration demonstrating a post-cAMP defect i steroidogenesis. Addition of the hydroxylated sterols, 20 or 25-OH cholesterol as substrate stimulated progesterone secretion in vehicle treated rats in a dose dependent fashion with 20-OH cholesterol being more potent. Progesterone secretion in response to stimulation with luteinizing hormone (LH) and cAMP from vehicle treated rats was less than that observed with 20 or 25-OH cholesterol, indicating that endogeneous susbtrate may be a limiting factor in steroid synthesis. The maximal capacity of luteal tissue to produced progestins following PGF 2α administration was determined with 20-OH cholesterol as the substrate. The results suggest that the post-cAMP defect at 4 hours following PGF 2α administration may be due to failure of the cells to mobilize endogeneous cholesterol. However at 24 hours following PGF 2α administration the decreased ability of luteal cells to convert cholesterol to pregnenolone may contribute to decreased progesterone synthesis.