In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.