Background: We have investigated different models for osteoblastic lesions. Currently, there are two models using MatLyLu R-3327prostate cancer cells: tumor cell application in the left heart ventricle and intravenous application with concomitant transient surgical clamping of the lower caval vein. Materials and Methods: Thirty male Copenhagen rats (age 9±2 months, mean weight 323±21 g) were each injected with 200,000 R-3327 prostate cancer cells. In 10 rats the left ventricle route was used (group 1), in an other 10 rats the intravenous route (group 2), while in the third group of 10 rats a new model of a direct intra-osseous route was applied (group 3). Additionally, a control group of 5 rats underwent the same procedure as in group 3, but only saline without tumor cells was administered. A ^99mTc-HMDP bone scan and histological examination of bone and lung were performed for follow-up. Results: In the bone scan, bone lesions could be not visualized in groups 1 and 2, but in group 3 osteoblastic lesions were observed in both femora in 9 out of 10 rats. Upon histological examination, there were lung metastases in animals from groups 1 and 2, but not in group 3. Clinical signs for bone metastases in the lumbar spine (motor disablement of the hind legs) were found in groups 1 and 2. Conclusion: The intra-osseous administration of MatLyLu R-3327 prostate cancer cells represents a useful and effective model for osteoblastic bone lesion, and allows further autoradiographic evaluation of bone uptake using bone-seeking radiopharmaceuticals.