During the peri-implantation period significant production of ovine interferon-tau (olFNtau) by the trophectoderm is detected in day 13-16 conceptuses, but its level rapidly declines thereafter. To understand molecular mechanisms by which oIFNtau gene expression is down-regulated, a variety of deletion constructs were prepared from upstream sequences of the oIFNtau gene and examined for possible silencer regions by using transient transfection into human choriocarcinoma, JEG3, cells. Two regions between -700 to -654 bases (distal region) and from -503 to -453 bases (proximal region) were found to be the possible negative regulatory regions. With probes prepared from these regions, gel mobility shift assay (GMSA) was then conducted. DNA-protein complexes were observed, but the gel shift pattern was different between nuclear extracts from days 14 (active oIFNtau production) and 20 (minute oIFNtau production) ovine trophoblasts. Day 20 nuclear extracts exhibited more band patterns than those of day 14; most notably the distal region between -692 and -668 bases exhibited the distinct band with nuclear extracts from day 20, but not from day 14 trophoblasts. In addition, the band patterns from day 20 trophoblast nuclear proteins were similar to those detected with JEG3 and HeLa cell nuclear extracts. Taken together, these observations suggest that the upstream sequences identified could serve as negative regulatory regions to which various nuclear factors bind, resulting in reduction of oIFNtau gene transcription.