Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir.Methods: Macaque rings containing 25 mg dapivirine, 100 mg dapivirine, 300 mg darunavir or 100 mg dapivirine + 300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC 50 values were calculated.Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present and the release medium. In macaques, serum concentrations of both microbicides were maintained between 10 1 and 10 2 pg/mL. Vaginal fluid levels ranged between 10 3 and 10 4 ng/g and between 104 and 10 5 ng/g for dapivirine and darunavir, respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8×10 3 -3.8×10 3 ng/g) .cervix (9.4×10 1 -3.9×10 2 ng/g) . uterus (0 -108 ng/g) .rectum (0 -40 ng/g). Measured IC 50 values were .2 ng/mL for both compounds.Conclusions: Based on these results, and in light of recent clinical progress of the 25 mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate.