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To evaluate the effect of the menstrual cycle in HIV-positive women on plasma and genital cytokine levels, interrelationships between vaginal and plasma cytokines, CD4 and CD8 T cell fluctuations, and genital and plasma viral loads.Plasma and cervicovaginal lavage specimens were collected from 55 HIV-positive women with CD4 cell counts < 350 cells/μl during phases of the menstrual cycle. Samples were assayed for IL-1β, IL-6, IL-4, IL-8, IL-10, TGFβ, TNFα, INFγ, MIP1α, MIP1β, RANTES, and TNFR-II using enzyme-linked immunosorbent assays. CD4 and CD8 T cell expression was evaluated by flow cytometry. Repeated measures regression models were used to assess the effect of the menstrual cycle on cytokines and viral load. Multivariate repeated regression models were used to assess the correlation among selected cytokines and between selected cytokines and HIV viral load.Vaginal IL-1β, IL-4, IL-6, IL-8, IL-10, MIP1β, RANTES, TGFβ, and TNFR-II were significantly elevated during menses but were not altered during other phases. Plasma cytokine levels were not altered during the menstrual cycle. A positive Candida culture increased vaginal IL-8 during menses, whereas vaginal discharge was associated with a reduction in vaginal IL-4, IL-10, and RANTES. CD4 and CD8 cell numbers did not vary with the menstrual cycle. Vaginal cytokine levels correlated only with vaginal viral load, in a sampling method-dependent manner.We provide evidence of elevated vaginal cytokine levels during menses, which appear to regulate vaginal and not plasma HIV shedding, suggesting that a menstrual cycle pattern exists for cytokine production in HIV-positive women impacting vaginal shedding of HIV.