The results of repeated human papillomavirus (HPV) DNA testing were compared to changes in cervical pathology and the composition of vaginal microorganisms. A cohort of 19 women with HPV cervical infections in the absence of cervical intraepithelial neoplasia at enrollment was reexamined on average at 7.3-month intervals over a 2-year period. At each follow-up visit, cytological and colposcopic examinations were done and vaginal microorganisms were assessed quantitatively by Gram staining of secretions, and anaerobic and aerobic culture. HPV genotypes 6, 11, 16, and 18 were detected by polymerase chain reaction analysis using DNA isolated from exfoliated cervical cells. The detection of HPV DNA was significantly associated with carriage of Grade II flora (P < 0.001), isolation of Gardnerella vaginalis (P = 0.03), Ureaplasma urealyticum (P = 0.04), Candida albicans (P = 0.01), Bacteroides species (P = 0.01), and overgrowth by anaerobes (P = 0.004). Normal vaginal flora, characterised by the predominance of Lactobacillus species, was significantly associated (P < 0.001) with a negative HPV test. The detection of HPV DNA is associated with the composition of microorganisms present in the vagina at the time of testing.