Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2–12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p > 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p < 0.05) with a 24-h pulse of dibutyryl adenosine 3′, 5′-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3–12) resulted in continuously stimulated (p < 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p < 0.05) percentage of cells staining positive for 3 β-hydroxy-Δ5-steroid dehydrogenase-Δ5, Δ4-isomerase (3β HSD) activity were recovered on Day 12 in cultures incubated (Days 3–12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2α(PGF2α) produced dose-dependent inhibition (p < 0.05) of progesterone secretion. Reduced numbers of 3β HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2α) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.