Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.