To study the vaginal flora of pregnant women at 22-28 weeks gestation to determine whether the presence of specific micro-organisms is significantly associated with preterm birth and prelabour rupture of the membranes. A comprehensive descriptive prospective study of the vaginal micro-flora of women between 22-28 weeks gestation comparing those who gave birth preterm (less than 37 weeks) with those who gave birth at term. Microbiological assessment included cultures for aerobic and anaerobic bacteria, yeasts, genital mycoplasmas and Trichomonas vaginalis. Multiple logistic regression analysis was used to account for confounding obstetric and demographic variables. The Queen Victoria Hospital, Adelaide, South Australia. 135 women who gave birth preterm compared to 651 women who gave birth at term. Preterm birth and preterm prelabour rupture of membranes (PROM) RESULTS: The prevalence of Gardnerella vaginalis between 22-28 weeks was significantly higher in women who gave birth preterm compared to women who gave birth at term (23% vs 15%; multiple logistic regression odds ratio (OR) 1.8, 95% confidence intervals (CI) 1.01-3.2, P less than 0.05. Ureaplasma urealyticum was also found in a higher proportion of women who gave birth preterm (49% vs 32% OR 1.7, 95% CI 1.1-2.6, P less than 0.0005). Preterm PROM occurred in 42% of whom 60% were carriers of U. urealyticum between 22-28 weeks, compared with 32% in the term group (OR 3.2, CI 1.7-6.1, P less than 0.0005). When women who received antibiotics between the midtrimester swab and labour were excluded, G. vaginalis was also significantly associated with preterm PROM (OR 2.7, CI 1.1-6.5, P less than 0.05). The presence of vaginal enteropharyngeal bacteria (E. coli, Klebsiella spp., Haemophilus spp., Staph. aureus) in the midtrimester was not predictive of preterm birth, but when these organisms were found in labour, they appeared to have been acquired later in the pregnancy. Women carrying G. vaginalis or U. urealyticum during the midtrimester had nearly twice the risk of preterm birth, while women positive for U. urealyticum had more than a threefold risk of preterm PROM.