OBJECTIVE Receptive anal intercourse in both men and women is associated with the highest probability for sexual acquisition of HIV infection. As part of a program to develop an effective prevention strategy, we performed an ex-vivo preclinical evaluation to determine the efficacy of multiple double combinations of maraviroc (MVC) and reverse transcriptase inhibitors (RTIs). DESIGN The entry inhibitor, MVC, a nucleotide RTI, tenofovir and two non-nucleoside RTIs, UC781 and TMC120 (dapivirine, DPV), were used in double, combinations against a panel of CCR5-using clade B and clade C HIV-1 isolates and against MVC-escape variants. A gel-formulated version of MVC-DPV combination was also tested. METHODS Indicator cells, cocultures of immature dendritic cells with CD4T cells, and colorectal tissue explants were used to assess antiviral activity of drug combinations. RESULTS All dual MVC-RTI combinations tested inhibited MVC-sensitive and resistant isolates in cellular and colorectal explants models. All the combinations were positive with no reduction in the activity of MVC. In tissue explants, the combinations against all viral isolates tested produced an increase in the activity of MVC. An initial gel-formulation of MVC-DPV combination showed greater and prolonged antiviral activity of MVC in mucosal tissue explants. CONCLUSION This study demonstrates that combinations based on antiretroviral drugs inhibiting HIV transmission at viral entry and reverse transcription have potential as prevention strategies against colorectal transmission of HIV-1 including MVC-resistant isolates. Preclinical evaluation with colorectal tissue explants indicates that a gel-formulation of MVC-DPV is an effective candidate colorectal microbicide.