Calcitonin gene–related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell–based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo–expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted β-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo–expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP–transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP–transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 ± 0.4 pmol CGRP/1 × 106 cells/48 hours (mean ± standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP–transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo–expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell–based cell and gene therapy strategy for the treatment of cardiovascular diseases.