Replication competent recombinant simian-human immunodeficiency virus encoding the reverse transcriptase gene (RT SHIV) from HIV-1 was characterized for vaginal transmission in rhesus macaques. RT SHIV was shown to transmit efficiently via the vaginal route in macaques with detectable plasma viremia persisting for a year in some animals. Analyses of virus load in tissues of infected animals revealed accumulation of viral RNA in lymph nodes and spleen with levels correlating with plasma viremia. RT-SHIV was inhibited by dapivirine, nevirapine, efavirenz and tenofovir in vitro, although the effect was less pronounced with tenofovir. Virus isolated from infected animals at early and later time points had limited changes in RT sequences and exhibited similar sensitivity to RT inhibitors as the challenge virus. The vaginal transmission of RT SHIV demonstrated here suggests this virus may possibly be used in the nonhuman primate model for limited evaluation of RT inhibitors applied vaginally.