OBJECTIVE: To evaluate the relationship between fetal fibronectin and preterm birth and maternal-to-child transmission of human immunodeficiency virus (HIV) in an African population of predominantly HIV-infected women. METHODS: During a trial of second trimester and intrapartum antibiotics compared with placebo to prevent chorioamnionitis and reduce preterm birth and mother-to-child transmission of HIV, vaginal fluid was collected before antibiotics (20-24 weeks) and after treatment at 28 weeks and assayed for fetal fibronectin. Pregnancy outcomes of 2,353 women delivering liveborn singleton infants are presented. RESULTS: Positive fetal fibronectin assays (50 ng/mL or more) were detected in 4.2% and 4.9% of samples at 20-24 weeks and 28 weeks. Positive fetal fibronectin assays at 28 weeks but not at 20-24 weeks were associated with lower mean birthweight (199 g, P<.001); lower mean gestational age (2 weeks, P<.001); six-fold higher rate of preterm birth less than 32 weeks (10.8% compared with 1.9%, odds ratio 6.3, 95% confidence interval 3.2-12.3) and a two-fold higher rate of preterm birth less than 37 weeks (38.7 compared with 22.0%, odds ratio 2.3, 95% confidence interval 1.5-3.3). Also, at 28 weeks, as the fetal fibronectin values increased, each of the outcomes worsened, and every test of trend was significant. An association between elevated fetal fibronectin levels and mother-to-child transmission of HIV was present at 20 to 24 weeks but not at 28 weeks. Antibiotic treatment at 20 to 24 weeks was not associated with fetal fibronectin levels at 28 weeks. CONCLUSION: In a population of predominantly HIV- infected African women, fetal fibronectin concentrations at 28 but not at 20-24 weeks were associated with increased risk of preterm birth. The associations were stronger for early preterm birth and when fetal fibronectin levels were higher. High levels of fetal fibronectin were positively associated with mother-to-child transmission of HIV at 20 -24 but not at 28 weeks. Antibiotic treatment did not influence fetal fibronectin levels. © 2007 The American College of Obstetricians and Gynecologists.