To monitor the bacterial vaginal microflora of postmenopausal women, several approaches were used: (1) Nugent scoring of Gram-stained smears from vaginal swabs; (2) testing for bacteria, using specific polymerase chain reaction (PCR) primers; (3) PCR-denaturing gradient gel electrophoresis (DGGE) analysis of total bacterial DNA, amplified using eubacterial and Lactobacillus-specific primers; and (4) sequence analysis of amplified DNA fragments of interest from denaturing gradient gels. Of 20 women studied, 70% had either intermediate-grade bacterial colonization or bacterial vaginosis (BV), as defined by Nugent criteria, at the first sampling point. Subjects with normal Nugent scores had >/=1 species of Lactobacillus detected by sequencing of dominant DNA fragments from denaturing gradient gels generated using eubacterial primers. Samples from women with Nugent scores that indicated BV had varied bacterial DGGE profiles, which emphasizes the diverse nature of such infections. This study indicates that asymptomatic BV appears to be much more common than is currently perceived; these findings may have implications for the health of postmenopausal women.