In ruminants, conceptus IFNT production is necessary for maintenance of pregnancy. We examined the role of PKA in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although over-expression of the catalytic subunit of PKA or the addition of 8-bromo-cyclic AMP had little ability to up-regulate boIFNT1 reporter constructs on their own, co-expression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between -126 and -67, which lacks a cAMP response element (CRE) but contains the functional Ets2-binding site and an AP1 site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1 binding site adjacent to the Ets2 site or pharmacological inhibition of MEK2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the co-activators, CBP/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 co-activation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2 and CBP/p300 and triggered via growth factors released from maternal endometrium.