We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.