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Protein kinase C (PKC) activity and its effect on progesterone production were investigated using porcine large and small luteal cells (LLC and SLC). Corpora lutea (CL) were surgically collected from pigs on Day 10 of the estrous cycle (Day 0 = onset of standing estrus). Luteal cells were dissociated by collagenase; LLC and SLC were further separated on a discontinuous Ficoll gradient. In a dose-response experiment with phorbol 12-myristate 13-acetate (PMA, a stimulator of PKC), progesterone production was not affected by 0.01 and 0.1 μM PMA, but was stimulated by 1 μM PMA. In a time series experiment, progesterone secretion was increased by 1 and 10 μM PMA in LLC by 60-150 min, and by 1 μM PMA in SLC during 120 and 150 min of incubation. However, 4α-phorbol ester did not affect progesterone synthesis. H-7, a PKC inhibitor, blocked PMA-stimulated progesterone secretion by LLC during 3 hr of incubation.Of the PKC activators tested at 10 μM, PMA significantly stimulated cytosolic PKC activity over that of natural PKC activators in both LLC and SLC, whereas 4α-phorbol ester did not affect PKC activity. H-7 inhibited PMA-stimulated PKC activity. PS (1-phosphatidyl-L-serine) + CA++ and PS + DG (1,2-dioleoyl-sn-glycerol) + Ca++ stimulated PKC activity. The results demonstrate that activation of PKC can increase progesterone secretion by porcine luteal cells from Day 10 of the estrous cycle and suggest PKC can have multiple effects in regulating luteal function.