Interferon-τ (IFNτ), produced by the trophectoderm of peri-implantation conceptuses in ruminant ungulates, attenuates the uterine production of a luteolytic factor, prostaglandin F 2α, resulting in the maintenance of corpus luteum function. However, molecular mechanisms regulating the temporal/spatial expression of IFNτ gene are not clearly understood. The 5′-upstream region of the sheep IFNτ (oIFNτ) gene was examined for its transcriptional regulation in two different cell types; JEG3 cells supported the transactivation of oIFNτ-reporter construct, but HeLa cells did not. In a heterologous SV40 enhancer–oIFNτ promoter or oIFNτ enhancer–SV40 promoter systems, elements required for such cell specific transactivation were localized between −654 and −555 bases, the enhancer, but not the basal promoter region of the oIFNτ gene. In these combinations, high degrees of transactivation were observed in JEG3 cells and the activity was further enhanced by the addition of phorbol 12-myristate 13-acetate (PMA), while those responses were absent in HeLa cells. To identify nucleotide sequences responsible for cell specific expression, transient transfection studies with sequential point mutations in the enhancer elements were executed. Transactivation of oIFNτ enhancer-reporter constructs was primarily regulated by three regions containing AP-1 site, GATA like sequence and site(s) unidentified. In gel mobility shift assays (GMSAs), the AP-1 site located in the enhancer region was recognized by nuclear extracts from both cell types. However, one of the GMSA probes containing GATA-like sequence exhibited different DNA-protein complex patterns in JEG3 and HeLa cells. Observations, in which the same upstream sequence behaved differently due possibly to kinds of nuclear factors available in these cell lines, suggest that such a sequence may be involved in cell specific transactivation of the oIFNτ gene. Furthermore, the same enhancer sequences were also recognized by nuclear extracts from sheep trophoblasts, suggesting that the enhancer sequences between −654 and −555 bases of oIFNτ gene may be functioning in vivo.