We previously reported that urokinase (uPA) is produced by the human prostate cancer cell line, PC-3, and could function as a growth factor for cells of the osteoblast phenotype. To examine the role of uPA in metastasis to the skeleton and to extraskeletal sites, we have developed a homologous model of uPA overexpression in a rat prostate cancer cell line. Full length cDNA encoding rat (r) uPA was isolated and subcloned as a 1.4-kilobase XbaI-BspHI fragment in the sense and antisense orientation into the Moloney murine leukemia retroviral vector pYN. The control (pYN) and experimental (pYN-ruPA, pYN-ruPA-AS) plasmids were transfected into Dunning R 3227, Mat LyLu rat prostate carcinoma cells. Experimental clones expressing at least 5-fold higher (pYN-ruPA) or 3-fold lower (pYN-ruPA-AS) than controls were selected, and control and experimental cells were inoculated into the left ventricles of inbred male Copenhagen rats. Animals were sacrificed at timed intervals to examine the evolution of metastatic lesions. Control animals developed metastases to the lumbar vertebrae resulting in spinal cord compression and hind limb paralysis at 20-21 days postinoculation. Animals inoculated with cells overexpressing uPA developed hind limb paralysis significantly earlier (by day 14-15 postinoculation). Additionally, more widespread skeletal (ribs, scapula, and femora) metastases were seen. Serum from experimental animals showed a progressive elevation in alkaline phosphatase levels, and histological examination of lumbar metastases revealed markedly increased osteoblastic activity over that observed in control animals. In contrast to this, animals inoculated with cells underexpressing uPA developed hind limb paralysis significantly later (days 25-29 postinoculation) and displayed decreased tumor metastasis. These studies support a role for the catalytic domain of uPA in enhancing both skeletal and nonskeletal prostate cancer invasiveness and are consistent with a role for the growth factor domain of uPA in mediating an osteoblastic skeletal response.