Neutral endopeptidase (NEP) is a cell surface peptidase that catalytically inactivates a variety of physiologically active peptides including basic fibroblast growth factor (FGF-2). We investigated the effect of using lentivirus to overexpress NEP in NEP-deficient DU145 prostate cancer cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP), catalytically inactive mutant NEP (L-NEPmu), and green fluorescent protein (L-GFP) were stably introduced into DU145 cells. FGF-2 levels in cell culture supernatants decreased by 80% in L-NEP-infected DU145 cells compared to cells infected with L-NEPmu or L-GFP (P<0.05) while levels of other angiogenic factors were not altered. In vitro tubulogenesis of human vascular endothelial cells induced by conditioned media from DU145 cells infected with L-NEP was significantly reduced compared with that from DU145 cells infected with L-GFP (P<0.05). Tumor xenografts from L-NEP-infected DU145 cells were significantly smaller compared to control cell xenografts and vascularity within these tumors was decreased (P<0.05). Our data suggest that stable expression of NEP in DU145 cells inhibits prostate cancer tumorigenicity by inhibiting angiogenesis, with a probable mechanism being proteolytic inactivation of FGF-2.