Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-τ on the release of prostaglandin F_2α (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-τ attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-τ attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-τ (50 or 500 ng/ml), PDBu rbIFN-τ, or PDBu PD98059 (MEK-1 inhibitor; 50 μM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-τ (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-τ. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-τ. Treatment of BEND cells with rbIFN-τ also did not attenuate PDBu-induced degradation of IκBα, suggesting that the IκBα/NFκB pathway is not a site of IFN-τ inhibition of PGF. However, rbIFN-τ did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-τ. Because rbIFN-τ inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-τ rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.