The relationship of genital flora assessed at the end of the second trimester of pregnancy and intraamniotic infection diagnosed by clinical signs and symptoms during labor was evaluated. Women were enrolled at 23–26 weeks of gestation and followed through delivery in the multicenter Vaginal Infections and Prematurity Study (1984–1989). Among the cohort of 11,989 followed through delivery, 286 (2.4%) developed intraamniotic infection. The recovery of Gardnerella vaginalis (relative risk [RR] = 1.8; 95% confidence interval [CI] = 1.4–2.4), heavy growth of Bacteroides species (RR = 1.5; 95% CI = 1.1–2.1), and isolation of Mycoplasma hominis (RR = 1.7; 95% CI = 1.3–2.1) from the vagina at the end of the second trimester of pregnancy were associated with an increased risk of intraamniotic infection. Bacterial vaginosis was also associated with intraamniotic infection (RR = 1.5; 95% CI = 1.1–2.2). These findings extend prior studies by showing that prenatal cultures for .microorganisms associated with bacterial vaginosis predicted an increased risk of intraamniotic infection.