BACKGROUND: Surrogate markers of HIV-1 pre-exposure prophylaxis and microbicide efficacy are needed. One potential surrogate is the antiviral activity in cervicovaginal lavage (CVL) after exposure to candidate products. We measured CVL antiviral activity in women using oral or vaginal tenofovir-based pre-exposure prophylaxis and correlated activity with drug and immune mediator levels. METHODS: Inhibitory activity against HIV-1 and herpes simplex virus (HSV)-2 and concentrations of interleukin (IL)-1β, IL-6, IL-8, interferon-γ, induced protein 10 (IP-10), macrophage inflammatory protein (MIP)-1α, MIP-3a, lactoferrin, secretory leukocyte protease inhibitor, and defensins were measured in CVL obtained from 60 women at baseline and after 6 weeks of a randomized sequence of oral and topical tenofovir. CVL tenofovir concentrations were measured by mass spectrometry. RESULTS: The number of women with CVL anti-HIV activity ≥90% increased significantly from 5.0% at baseline to 89.1% after daily use of 1% tenofovir gel (relative risk = 17.85, P < 0.001), but there was no increase after daily oral tenofovir. The CVL anti-HIV activity correlated with drug levels (Spearman correlation coefficient 0.64 after tenofovir gel; P < 0.001) but not with the concentrations of mucosal immune mediators. No increase in CVL anti-HSV activity was observed after either drug regimen, an observation consistent with the higher concentrations of tenofovir needed to inhibit HSV-2 infection. The CVL anti-HSV activity correlated with lactoferrin, defensins, IP-10, IL-8, and detectable levels of MIP-1α but not with drug levels. CONCLUSIONS: CVL may provide a surrogate for local but not systemic drug efficacy and a tool to better understand mucosal factors that modulate antiviral activity in genital tract secretions. © 2013 by Lippincott Williams and Wilkins.