Cross-sectional studies have suggested that intravaginal practices, such as douching or "dry sex," may increase women's susceptibility to infection with human immunodeficiency virus (HIV). The authors examined the temporal nature of this association in a cohort of South African women. At enrollment (2001-2002), 4,089 women were tested for HIV infection. Participants reported their intravaginal practices at a 6-month follow-up visit and were followed with repeat HIV testing for up to 24 months. Among the 3,570 women who were HIV-negative at enrollment, 26% reported some type of intravaginal practice, mostly washing inside the vagina with water and/or cloth as part of daily hygiene. During follow-up, 85 incident HIV infections were observed. Intravaginal practices were associated with prevalent HIV at enrollment (adjusted odds ratio = 1.50, 95% confidence interval: 1.22, 1.85), but during follow-up there was no association between intravaginal practices and incident HIV (adjusted hazard ratio = 1.04, 95% confidence interval: 0.65, 1.68). These findings may be explained by a reversal of the causal sequence assumed for this association, since intravaginal practices may be undertaken in response to vaginal infections that occur more commonly among HIV-infected women. Intravaginal practices appear unlikely to be a cofactor in the male-to-female transmission of HIV in this setting.