Gastrointestinal nonepithelial tissue is a useful target for in vivo gene transfer. The aim of this study was to investigate whether gene transfer into this organ could be achieved by submucosal injection of plasmid DNA. Plasmid DNA carrying either the firefly luciferase or Escherichia coil LacZ reporter gene was injected directly into the gastric submucosa of adult rats. Gene expression was characterized by quantitative luciferase assay and qualitative in situ β-galactosidase (β-Gal) staining. Luciferase activity was detected as early as 1 day after injection, increased markedly at 2 days, and then decreased. Some of the rats showed detectable levels of luciferase expression at 14 and 21 days postinjection. Histochemical staining for β-Gal demonstrated that expression of the recombinant genes was localized in smooth muscle cells of the muscularis mucosae and the muscular layer and mesenchymal cells in the lamina propria. Our results indicate that gene transfer into the gastrointestinal tract can be achieved by simple needle insertion of naked plasmid DNA into the submucosa.