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Determine the frequency of genital HIV-1 shedding in a large cohort of women on long-term suppressive antiretroviral therapy (ART) and its association with mucosal inflammation.We measured levels of HIV-1 RNA and inflammation biomarkers in cervicovaginal lavage (CVL) from HIV-seropositive women enrolled in the Women's Interagency HIV Study (WIHS).HIV-1 was quantified (Abbott RealTime HIV-1 assay) from CVL samples of 332 WIHS participants with and without clinical evidence of genital inflammation at the time of CVL collection; participants had suppressed plasma viral load (PVL; limit of quantitation less than 20-4000 copies/ml depending on year of collection) for a median of 7.1 years [interquartile range (IQR) 3.4–9.8, Group 1] or for a median of 1.0 years (IQR = 0.5–1.0, Group 2). Twenty-two biomarkers of inflammation were measured in CVL to compare with clinical markers.HIV-1 was detected in 47% of 38 pre-ART CVL samples (median 668 copies/ml) and detection in CVL was associated with higher pre-ART PVL. HIV-1 was detected in only 1 of 38 CVL samples from these women on suppressive antiretroviral therapy for 1 year. No HIV-1 RNA was detected in 294 CVL samples from a cross-sectional set of women with suppressed PVL for a median of 7 years. Clinical inflammation markers were correlated with inflammatory biomarkers in CVL specimens, although genital inflammation was not associated with measurable genital HIV-1 shedding in these WIHS participants on ART.ART that suppresses HIV-1 in the plasma of women also prevents genital tract HIV-1 shedding, even in the presence of genital tract inflammation.