Interferon stimulated gene 17 (ISG17) and Mx are up-regulated in the ruminant uterus in response to interferon-tau (IFNtau) during early pregnancy. Recent evidence strongly indicates that expression of ISGs occur only in stroma (ST) and glandular epithelium (GE) during this time as a result of transcriptional repression by interferon regulatory factor two (IRF-2) expression in the LE. The present report tested this hypothesis by examining mRNA and protein expression of ISG17 and Mx in serial uterine cross-sections obtained from cyclic and early pregnant ewes. In situ and immunocytochemical analysis revealed that ISG17 mRNA and protein were low to undetectable, whereas Mx mRNA was expressed in the lumenal (LE) and superficial GE at all days of the estrous cycle examined. Both ISG17 and Mx mRNA increased in the stratum compactum ST between Days 11 and 13, and expression extended into the deep GE and stratum spongiosum ST on Days 15 through 17 in pregnant ewes. Interestingly the Mx gene continued to be strongly expressed in LE and superficial GE through Day 17 of pregnancy, whereas ISG17 remained low to undetectable in these cells. Collectively, this study highlights the complexity of the uterine environment by unequivocally illustrating differential temporal and spatial expression of the IFN-responsive genes ISG17 and Mx.