To determine the prevalence of Gardnerella vaginalis in women with normal vaginal flora. Women without symptoms or signs of vaginal infection and five or fewer lifetime sexual partners were recruited for a longitudinal study of vaginal flora. Negative Amsel criteria and a Nugent score of 0-3 were required for enrolment. Vaginal specimens were self-collected daily for Gram stain and every 3 days for PCR for G vaginalis for 30 days. Women completed daily diaries recording sexual activity, symptoms and menses. Twenty women were recruited for the study with 19 completing all specimens and 1 lost to follow-up. During the 30-day study period, 13/19 (68.4%) of women had normal Nugent scores (0-3) whereas 6/19 (31.6%) of women had at least 2 days of Nugent scores in the intermediate range (p=0.09). Among the 19 women, 9 (47%) were negative for G vaginalis by PCR throughout the study period whereas 10 (53%) had at least one specimen that demonstrated the presence of G vaginalis by PCR. Of those women with intermediate flora on Gram stain during the course of the study 5/6 (83.3%) were positive for G vaginalis while 5/13 (38.5%) of those women with only normal Nugent scores were positive for G vaginalis. Thus, 61.5% of women with normal Nugent scores had no evidence of G vaginalis by serial PCR. Gardnerella may not be part of the normal flora in women with optimal vaginal health.