Objective: We sought to determine the range of fetal fibronectin values in the vagina from 8 to 22 weeks’ gestation, the factors associated with both low and high values, and whether high values are associated with gestational age at birth. Study Design: Vaginal fetal fibronectin was quantitatively determined in a prospective cohort study of 13,360 women being evaluated for participation in the National Institute of Child Health and Human Development Maternal-Fetal Medicine Unit treatment trials for bacterial vaginosis and Trichomonas vaginalis. Fetal fibronectin values were correlated with gestational age at screening, race, the presence of bacterial vaginosis and Trichomonas vaginalis, and gestational age at delivery. Results: Vaginal fetal fibronectin values at each gestational age ranged from unmeasurable to >1000 ng/mL, with median values always being <10 ng/mL. Fetal fibronectin values declined progressively with increasing gestational age at sampling. Bacterial vaginosis and black race were associated with higher values, whereas nulliparity was associated with lower values. High values after 13 weeks’ gestation were associated with a 2- to 3-fold increased risk of subsequent spontaneous preterm birth overall and a 4-fold increased risk of very early preterm birth. Conclusion: Elevated vaginal fetal fibronectin levels from 13 to 22 weeks’ gestation are associated with a significantly increased risk of spontaneous preterm birth. (Am J Obstet Gynecol 2000;183:469-75.)