In order to investigate the antitumor and antimetastatic efficacy of new chemotherapeutic agents, a novel, red-fluorescent, orthotopic model of pancreatic cancer was constructed in nude mice. MIA-PaCa-2 human pancreatic cancer cells were transduced with red fluorescent protein (RFP) and initially grown subcutaneously. Fluorescent tumor fragments were then transplanted onto the pancreas by surgical orthotopic implantation (SOI), facilitating high-resolution, real-time visualization of tumor and metastatic growth and dissemination in vivo. Tumor growth at the primary site was visible within the first postoperative week, while distant metastasis and the development of ascites became visible over the following week. This MIA-PaCa-2-RFP model produced extensive local disease and metastases to the retroperitoneum (100%), spleen (100%), intestinal and periportal lymph nodes (100%), liver (40%) and diaphragm (80%), and gave rise to malignant ascites and peritoneal carcinomatosis in 80% of cases. Growth and metastasis of tumor was more rapid and frequent than in previously described orthotopic pancreatic cancer models, leading to a median survival of only 21 days after tumor implantation. This unique, red fluorescent model rapidly and reliably simulates the highly aggressive course of human pancreatic cancer and can be easily non-invasively visualized in the live animal. The model can therefore be used for the discovery and evaluation of novel therapeutics for the treatment of this devastating disease.