When Lactobacillus spp. dominate the vaginal microbiota of women of reproductive age they acidify the vagina to pH /=0.4 (w/w) l-lactic acid. At a typical vaginal pH of 3.8, l-lactic acid at 1 (w/w) more potently and rapidly inactivated HIVBa-L and HIV-1 transmitter/founder strains compared with 1 (w/w) acetic acid and with acidity alone, all adjusted to pH 3.8. A final concentration of 1 (w/w) l-lactic acid maximally inactivated HIVBa-L in the presence of cervicovaginal secretions and seminal plasma. The anti-HIV activity of l-lactic acid was pH dependent, being abrogated at neutral pH, indicating that its virucidal activity is mediated by protonated lactic acid and not the lactate anion. CONCLUSIONS: l-lactic acid at physiological concentrations demonstrates potent HIV virucidal activity distinct from acidity alone and greater than acetic acid, suggesting a protective role in the sexual transmission of HIV.