SPL7013 Gel (VivaGel((R))) is a microbicide in development for prevention of HIV and HSV. This clinical study assessed retention and duration of antiviral activity following vaginal administration of 3 SPL7013 Gel in healthy women. Participants received 5 single doses of product with >/=5 days between doses. A cervicovaginal fluid (CVF) sample was collected using a SoftCup pre-dose, and immediately, or 1, 3, 12 or 24 h post-dose. HIV-1 and HSV-2 antiviral activities of CVF samples were determined in cell culture assays. Antiviral activity in the presence of seminal plasma was also tested. Mass and concentration of SPL7013 in CVF samples was determined. Safety was assessed by reporting of adverse events. Statistical analysis was performed using the Wilcoxon signed-rank test with Bonferroni adjustment; p90 of initial HIV-1 and HSV-2 inhibition was maintained in 6/11 women. SPL7013 was recovered in CVF samples obtained at baseline (46 of 105 mg dose). At 3 and 24 h, 22 mg and 4 mg SPL7013, respectively, were recovered. More than 70 inhibition of HIV-1 and HSV-2 was observed if there was >0.5 mg SPL7013 in CVF samples. High levels of antiviral activity were retained in the presence of seminal plasma. VivaGel was well tolerated with no signs or symptoms of vaginal, vulvar or cervical irritation reported. Potent antiviral activity was observed against HIV-1 and HSV-2 immediately following vaginal administration of VivaGel, with activity maintained for at least 3 h post-dose. The data provide evidence of antiviral activity in a clinical setting, and suggest VivaGel could be administered up to 3 h before coitus. TRIAL REGISTRATION: The study is registered at ClinicalTrials.gov under identifier: NCT00740584.