ABSTRACT Vaginally applied microbicides hold promise as a strategy to prevent sexual HIV transmission. Several nonspecific microbicides, including the polyanion cellulose sulfate, have been evaluated in large-scale clinical trials but have failed to show significant efficacy. These findings have prompted a renewed search for preclinical testing systems that can predict negative outcomes of microbicide trials. Moreover, the pipeline of potential topical microbicides has been expanded to include antiretroviral agents, such as reverse transcriptase, fusion, and integrase inhibitors. Using a novel ex vivo model of vaginal HIV-1 infection, we compared the prophylactic potentials of two forms of the fusion inhibitor T-20, the CCR5 antagonist TAK-778, the integrase inhibitor 118-D-24, and cellulose sulfate (Ushercell). The T-20 peptide with free N- and C-terminal amino acids was the most efficacious compound, causing significantly greater inhibition of viral genomic integration in intraepithelial vaginal leukocytes, measured by an optimized real-time PCR assay, than the more water-soluble N-acetylated T-20 peptide (Fuzeon) (50% inhibitory concentration [IC ₅₀ ], 0.153 μM versus 51.2 μM [0.687 ng/ml versus 230 ng/ml]; P < 0.0001). In contrast, no significant difference in IC ₅₀ s was noted in peripheral blood cells (IC ₅₀ , 13.58 μM versus 7.57 μM [61 ng/ml versus 34 ng/ml]; P = 0.0614). Cellulose sulfate was the least effective of all the compounds tested (IC ₅₀ , 1.8 μg/ml). These results highlight the merit of our model for screening the mucosal efficacies of novel microbicides and their formulations and potentially rank ordering candidates for clinical evaluation.