OBJECTIVE: Our objective was to determine any associations among midtrimester cervical fluid sialidase activity, bacterial vaginosis, and subsequent spontaneous preterm birth. STUDY DESIGN: In this nested case- control study all patients (n = 126) with spontaneous preterm birth at <35 weeks' gestation and selected control subjects delivered at ≥37 weeks' gestation (n = 126, matched for race, parity, and center) were derived from women enrolled in the multicenter National Institute of Child Health and Human Development Preterm Prediction Study. Sialidase activity and presence of bacterial vaginosis according to Gram stain were determined in cervical swabs and vaginal smears, respectively, obtained at 22 weeks' to 24 weeks 6 days' gestation. RESULTS: The mean±SD sialidase activities were similar in case patients and control subjects (0.64±1.60 vs 0.41±0.94 nmol·mL-1·min-1, P = .21). Neither sialidase activity above the 90th percentile (10.3% vs 9.5%, P = .8) nor sialidase activity above the 95th percentile (7.9% vs 4.8%, P = .3) of control specimens (>1.43 and >2.23 nmol·mL-1·min-1, respectively) was associated with spontaneous preterm birth. The frequency of combinations of bacterial vaginosis and elevated sialidase activity was similar (P≥.63 with either cutoff) in case patients and control subjects. Sialidase activity was significantly higher among women with bacterial vaginosis than among those without bacterial vaginosis (1.35±1.87 vs 0.03±0.14 nmol·mL-1·min-1, P<.0001). CONCLUSIONS: Elevated cervical fluid sialidase activity at 22 to 24 weeks' gestation did not distinguish women at increased risk for spontaneous preterm birth, nor did it discriminate a subgroup of patients who had bacterial vaginosis associated with spontaneous preterm birth.