Preterm birth, the major cause of neonatal mortality in developed countries, is associated with intrauterine infections and inflammation, although the exact mechanisms underlying this event are unclear. In this study, we show that circulating fetal DNA, which is elevated in pregnancies complicated by preterm labor or preeclampsia, triggers an inflammatory reaction that results in spontaneous preterm birth. Fetal DNA activates NF-kappaB, shown by IkappaBalpha degradation in human PBMCs resulting in production of proinflammatory IL-6. We show that fetal resorption and preterm birth are rapidly induced in mice after i.p. injection of CpG or fetal DNA (300 mug/dam) on gestational day 10-14. In contrast, TLR9(-/-) mice were protected from these effects. Furthermore, this effect was blocked by oral administration of the TLR9 inhibitor chloroquine. Our data therefore provide a novel mechanism for preterm birth and preeclampsia, highlighting TLR9 as a potential therapeutic target for these common disorders of pregnancy.