The biological potential of tumor cells is best evaluated at the organ site orthotopic to the tumor cells. Recent studies have documented site-specific differences in the potential of tumor cell growth. However, orthotopic implantation of bladder cancer cells into bladders of nude mice only resulted in a low tumor yield. We have developed a new model that consists of a rat bladder transplanted into the retroperitoneal space and connected to a reservoir s.c. placed in a nude mouse. Rat malignant bladder cancer cells (MYU3L and LMC19) transfected with the human growth hormone (hGH) gene as a biomarker were introduced into the transplanted bladder by percutaneous puncture of the attached reservoir. Successful uptake was indicated by a progressive rise in the hGH level in the bladder aspirate. When examined at 6-16 weeks post transplantation, all mice that had received MYU3L (n = 6) or LMC19 (n = 6) cells were found to have invasive carcinomas. MYU3L was highly invasive, forming multiple peritoneal implants, but was not metastatic. LMC19 was deeply invasive and metastasized to the retroperitoneal and subclavian lymph nodes and the lungs (4/6). Of two human bladder cancer cell lines (RT4 and T24) tested, RT4 formed multiple minute papillary tumors in five of six bladders, two of which were minimally invasive to the muscle layer. T24 cells formed only one to two small tumors in three of six bladders, and these were confined to the lamina propria. This system appears promising for studies of the mechanism of tumor invasion and metastasis and for evaluation of antineoplastic agents.