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Women of reproductive age with a Lactobacillus-dominated vaginal microbiota have a reduced risk of acquiring and transmitting HIV and a vaginal pH of ~4 due to the presence of ~1% (wt/vol) lactic acid. While lactic acid has potent HIV virucidal activity in vitro, whether lactic acid present in the vaginal lumen inactivates HIV has not been investigated. Here we evaluated the anti-HIV-1 activity of native, minimally diluted cervicovaginal fluid obtained from women of reproductive age (n = 20) with vaginal microbiota dominated by Lactobacillus spp. Inhibition of HIV_Ba-L was significantly associated with the protonated form of lactic acid in cervicovaginal fluid. The HIV_Ba-L inhibitory activity observed in the <3-kDa acidic filtrate was similar to that of the corresponding untreated native cervicovaginal fluid as well as that of clarified neat cervicovaginal fluid subjected to protease digestion. These ex vivo studies indicate that …