Superluteinized rats were injected with the prostaglandin F2 alpha (PGF2 alpha) analogue cloprostenol to induce luteolysis. The treatment decreased progesterone production of ovarian homogenates from 8.9 +/- 0.5 to 4.0 +/- 0.7 nmol/ovary/10 min (mean +/- SEM) within 40 min. tochondrial fractions isolated from control and cloprostenol treated animals produced 4.7 +/- 0.4 and 2.8 +/- 0.3 nmol progesterone/ovary/10 min, respectively. Thus, the PGF2 alpha analogue treatment significantly reduced mitochondrial progesterone production. Addition of the 15 000 X g supernatant fraction did not influence the progesterone production rates of the mitochondrial fraction. The basal progesterone secretion from quartered ovaries decreased from 1.50 +/- 0.15 to 0.38 +/- 0.05 nmol/ovary during the initial 15 min of incubation following cloprostenol administration. hCG and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBC) stimulated the progesterone secretion from quartered ovaries, but the response was delayed in ovaries obtained from cloprostenol treated animals. Although the response was delayed, the progesterone secretion following cloprostenol treatment was re-activated with cAMP either directly or via hCG. The increment in progesterone secretion above unstimulated controls in response to DBC was not influenced by the cloprostenol treatment while the increment caused by hCG was decreased. Our data suggest that: 1) PGF2 alpha deactivates mitochondrial progesterone production, 2) this deactivation may be overcome by cAMP, and 3) PGF2 alpha decreases gonadotrophin responsive adenylyl cyclase.