A longitudinal study was conducted to evaluate the viral shedding present in cervicovaginal secretions of HIV-1-seropositive women receiving antiretroviral therapy. A total of 128 paired cervicovaginal and blood samples was obtained from 37 women during a median follow-up period of 21 months. A sensitive, competitive, polymerase chain reaction and a reverse transcription polymerase chain reaction were used for the simultaneous quantitation of HIV-1 proviral DNA and RNA in cervicovaginal cells and cell-free RNA in cervicovaginal secretions, as well as HIV-1 RNA in peripheral blood. The cumulative probability of detecting proviral DNA in genital secretions was significantly higher over time in women with detectable viremia than in women in whom HIV-1 RNA was persistently undetectable in plasma (< 50 copies/ml) (P = 0.028 by log-rank test). The presence and amount of proviral DNA, cell-associated RNA and cell-free RNA in the cervicovaginal secretions were positively correlated with the presence of detectable viremia or the number of HIV-1 RNA copies in plasma (Spearman rank correlation, 0.290, 0.279, and 0.305, respectively; all P < 0.01), but no correlation was found with the CD4+ cell count. In addition, vaginal infections were positively correlated with the detection of proviral DNA in cervicovaginal secretions (odds ratio, 2.60; 95% confidence interval, 1.07-5.70). However, the positive correlation between the presence and amount of HIV in cervicovaginal secretions and the viral load in plasma provides no assurance that HIV shedding does not occur in the genital tract of women with undetectable HIV-RNA in plasma.