Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Previous gene transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have resulted in low-level expression for <1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity attributable to gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. We detected the transgene-derived Epo mRNA by reverse transcription polymerase chain reaction only in the kidneys injected with pCAGGS-Epo. After an injection of 100 mg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 +/- 71.8 mU/ml at week 5, and gradually decreased to 116.2 +/- 38.7 mU/ml at week 24. A similar pattern was obtained using smaller doses of plasmid, 2 μg or 30 μg of pCAGGS-Epo. Transgene-derived Epo secretion resulted in significant erythropoiesis. This novel technique is simple and safe, allowing high-level and long-term stable gene expression specific to the fibroblasts near the PTC, and should have therapeutic value for future applications in humans.