AbstractHorizontal gene transfer in bacteria is widely believed to occur via conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. Here, we report that cell wall-deficient bacteria can engulf DNA and other extracellular material via an endocytosis-like process. Specifically, we show that L-forms of the filamentous actinomycete Kitasatospora viridifaciens can take up plasmid DNA, polysaccharides (dextran) and 150-nm lipid nanoparticles. The process involves invagination of the cytoplasmic membrane, leading to formation of intracellular vesicles that encapsulate extracellular material. DNA uptake is not affected by deletion of genes homologous to comEC and comEA, which are required for natural transformation in other species. However, uptake is inhibited by sodium azide or incubation at 4 °C, suggesting the process is energy-dependent. The encapsulated materials are released into the cytoplasm upon degradation of the vesicle membrane. Given that cell wall-deficient bacteria are considered a model for early life forms, our work reveals a possible mechanism for primordial cells to acquire food or genetic material before invention of the bacterial cell wall.