The objective of this study was to examine genital tissue, vaginal fluid, and vaginal microbial flora at 3 phases of the menstrual cycle in asymptomatic women. Vaginal examinations were performed 3 times in 74 women: at the menstrual phase (days 1–5), the preovulatory phase (days 7–12), and the postovulatory phase (days 19–24). Flora of 50 women without bacterial vaginosis (BV) was analyzed separately from flora of 24 women with BV. The volume of vaginal discharge increased and the amount of cervical mucus decreased over the menstrual cycle. Among subjects without BV, the rate of recovery of any Lactobacillus changed little (range, 82% to 98%; P = .2); however, a small increase occurred in the rate of recovery of heavy (3+ to 4+ semiquantitative) growth of Lactobacillus over the menstrual cycle (P = .04). A linear decrease occurred in the rate of recovery of heavy growth of any non-Lactobacillus species, from 72% at days 1–5 to 40% at days 19–24 (P = .002). A linear decrease also occurred in the rate of recovery of Prevotella species, from 56% on days 1–5 to 28% on days 19–24 (P = .007), while a small linear increase occurred in the rate of recovery of Bacteroides fragilis (P = .05). Among subjects with BV, the only significant change was an increase in the rate of recovery of Lactobacillus, from 33% at days 1–5 to 54% at days 19–24 (P = .008). Among all subjects, the rate of recovery of heavy growth of Lactobacillus increased over the menstrual cycle and, in contrast, the concentration of non-Lactobacillus species tended to be higher at menses, which is evidence that the vaginal flora becomes less stable at this time.