Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long-term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1-based lentiviral vector encoding LacZ. Expression of beta-galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line-derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.