The present study was conducted to investigate whether arachidonic acid and its metabolites can modulate progesterone (P4) secretion in ovine chorionic cells. At concentrations of 7.5 μmol/l and 12.5 μmol/l, arachidonic acid caused an increase of basal P4 secretion (about 1.8-fold (p< 0.01) and 2.5-fold (p<0.001), respectively, over control). Such a stimulatory effect was suppressed when the concentration of arachidonic acid attained 25 μmol/l, and at 50 μmol/l the fatty acid led to a decline of basal P4 synthesis (about 35%, p <0.01). Phospholipase A2 (PLA2) and melittin had a similar dual effect to that observed when arachidonic acid was added exogenously. In contrast, eicosatrienoic acid (a closely related fatty acid) did not stimulate P4 secretion but inhibited it at a concentration of 50 μmol/l (about 40% inhibition, p <0.01). The possible involvement of calcium on the effects of arachidonic acid was explored. Interestingly, 3 mmol/l ethylene glycol bis(β-aminoethyl ether)-N,N,N,N′-tetraacetic acid (EGTA) and 10 μmol/l 8-N, N-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) further enhanced the steroidogenic effect of 12.5 μmol/l arachidonic acid (p<0.05 and p<0.01 vs the corresponding value in the absence of EGTA or TMB-8, respectively). In contrast, these agents failed to modify P4 secretion observed in the presence of 50 μmol/l arachidonic acid. We also tested the effect of inhibition of arachidonic acid metabolism via cyclooxygenase and lipoxygenase pathways. Indomethacin (10 μmol/l) failed to block the effects of arachidonic acid, but nordihydroguaiaretic acid (10 μmol/l) prevented the stimulatory action of this fatty acid. Taken together, these data suggest that arachidonic acid and its metabolites (perhaps its lipoxygenated metabolites) may be important intracellular regulators of P4 secretion in ovine chorionic cells.