Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for > or =21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases.