Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses.In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation andmaturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes tosubsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lungand blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and humanCpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lungmonocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro wasassessed by interleukin (IL)-6, tumor necrosis factor-a (TNF-a) and hydrogen peroxide. Monocytes from pretermcontrols had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- a and hydrogenperoxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- aand hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptorassociated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. Thesemodulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.