ABSTRACT: Aims: Exosomes are lipid bilayer vesicles of 50–100 nm released by basically all cell types. We recently reported that full-length HIV-1 RNA and lentiviral vector (LV) genome associate with exosomes through similar mechanisms. Here, we investigated the fate of lentiviral RNA shuttled by exosomes in target cells. Material & methods: Exosomes from cells transduced by a LV expressing green fluorescent protein under the control of an heterologous promoter were purified by iodixanol gradients and used to evaluate the LV expression in target cells. Results: The genome of LV incorporated in exosomes can be expressed in HIV-1-infected cells, but not in those that are uninfected, despite apparently similar levels of exosome internalization. The expression disappeared 2–3 days after challenge, and was blocked by pre-treatment with azidothymidine. Conclusion: Lentiviral genome incorporated in exosomes can be expressed in target cells having reverse transcriptase activity.