BACKGROUND Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important category of drugs for both chemotherapy and prevention of human immunodeficiency virus type 1 (HIV-1) infection. However, current non-human primate (NHP) models utilizing simian immunodeficiency virus (SIV) or commonly used chimeric SHIV (SIV expressing HIV-1 envelope) are inadequate due to the insensitivity to NNRTIs. To develop a NHP model for evaluation of NNRTI compounds, we characterized a RT-SHIV virus that was assembled by replacing the SIV mac239 reverse transcriptase (RT) with that of HIV-1HXB2. Since RT-SHIV exhibited in vitro characteristics of high infectivity, CCR5-usage, and sensitivity to HIV-1 specific NNRTIs, this virus was thought to be suitable for mucosal transmission and then was used to carry out a vaginal transmission study in pigtail macaques (Macaca nemestrina). RESULTS RT-SHIV exhibited in vitro characteristics of an infectious CCR5-tropic chimeric virus. This virus was not only highly sensitive to HIV-1 RT specific NNRTIs; its replication was also inhibited by a variety of NRTIs and protease inhibitors. For in vivo vaginal transmission studies, macaques were either pretreated with a single dose of DMPA (depot medroxyprogesterone acetate) or left untreated before intravaginal inoculation with 500 or 1,000 TCID50 of RT-SHIV. All macaques became systemically infected by 2 or 3 weeks post-inoculation exhibiting persistent high viremia, marked CD4+T cell depletion, and antiviral antibody response. DMPA-pretreated macaques showed a higher mean plasma viral load after the acute infection stage, highly variable antiviral antibody response, and a higher incidence of AIDS-like disease as compared with macaques without DMPA pretreatment. CONCLUSION This chimeric RT-SHIV has exhibited productive replication in both macaque and human PBMCs, predominantly CCR5-coreceptor usage for viral entry, and sensitivity to NNRTIs as well as other anti-HIV compounds. This study demonstrates rapid systemic infection in macaques following intravaginal exposure to RT-SHIV. This RT-SHIV/macaque model could be useful for evaluation of NNRTI-based therapies, microbicides, or other preventive strategies.