Frequent side effects of radical treatment modalities and the availability of novel diagnostics have raised the interest in focal therapies for localized prostate cancer. To improve the selectivity and therapeutic efficacy of such therapies, we developed a minimally invasive procedure based on a novel polymeric photosensitizer prodrug sensitive to urokinase-type plasminogen activator (uPA). The compound is inactive in its prodrug form and accumulates passively at the tumor site by the enhanced permeability and retention effect. There, the prodrug is selectively converted to its photoactive form by uPA, which is overexpressed by prostate cancer cells. Irradiation of the activated photosensitizer exerts a tumor-selective phototoxic effect. The prodrug alone (8 μmol/L) showed no toxic effect on PC-3 cells, but upon irradiation the cell viability was reduced by 90%. In vivo, after systemic administration of the prodrug, PC-3 xenografts became selectively fluorescent. This is indicative of the prodrug accumulation in the tumor and selective local enzymatic activation. Qualitative analysis of the activated compound confirmed that the enzymatic cleavage occurred selectively in the tumor, with only trace amounts in the neighboring skin or muscle. Subsequent photodynamic therapy studies showed complete tumor eradication of animals treated with light (150 J/cm(2) at 665 nm) 16 hours after the injection of the prodrug (7.5 mg/kg). These promising results evidence the excellent selectivity of our prodrug with the potential to be used for both imaging and therapy for localized prostate cancer.