We have investigated the interaction of Gluconacetobacter diazotrophicus, a non-nodulating endophytic nitrogen-fixing bacterium isolated from the intercellular spaces of sugarcane, with Arabidopsis thaliana and the crop plants maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), oilseed rape (Brassica napus), tomato (Lycopersicon esculentum), and white clover (Trifolium repens). Using seedlings grown aseptically in sucrose-containing culture media, we have shown that inoculation with very low numbers of G. diazotrophicus results in extensive intracellular colonization of root meristems and progressive systemic intracellular root colonization. Light microscopic examination of thin sections of resin-embedded root tips of Arabidopsis and these crop plants inoculated with β-glucuronidase (GUS)-labeled and with NifH promoter–GUS-labeled G. diazotrophicus showed blue-stained G. diazotrophicus within the cytoplasm of root cells, indicating that intracellular conditions were suitable for nitrogenase gene expression. Electron microscopy confirmed that these blue-stained intracellular G. diazotrophicus were within membrane-bounded vesicles. We discuss whether these novel inoculations with G. diazotrophicus are likely to enable non-nodular endosymbiotic nitrogen fixation and whether these inoculations can also provide a plant system to investigate the endosymbiotic theory of the origin of eukaryotic organelles.