To circumvent the problems of in vivo transfection and avoid the use of viral vectors or proteins, we sought to establish whether smooth-muscle cells (SMCs) transfected ex vivo could be delivered via the systemic venous circulation into the pulmonary bed to achieve local transgene expression in the lung. Primary cultures of pulmonary artery SMCs from Fisher 344 rats were labeled with a fluorescent, membrane-impermeable dye chloromethyl trimethyl rhodamine or transfected with the beta-galactosidase (betaGal) reporter gene under the control of the cytomegalovirus (CMV) enhancer/promoter (pCMV-beta). Transfected or labeled SMCs (5 x 10(5) cells/animal) were delivered to syngeneic recipient rats by injection into the jugular vein; the animals were killed at intervals between 15 min and 2 wk; and the lungs, spleens, kidneys, and skeletal muscle were excised and examined. At 15 min after transplantation, injected cells were detected mainly in the lumen of small pulmonary arteries and arterioles, often in groups of three or more cells. After 24 h, labeled SMCs were found incorporated into the vascular wall of pulmonary arterioles, and transgene expression persisted in situ for 14 d with no evidence of immune response. Using simple geometric assumptions, it was calculated that approximately 57 +/- 5% of the labeled cells reintroduced into the venous circulation could be identified in the lungs after 15 min, 34 +/- 7% at 48 h, 16 +/- 3% at 1 wk, and 15 +/- 5% at 2 wk. Similar results were observed using cells transfected with the reporter gene betaGal. To determine whether this method of gene transfer could prove effective in inhibiting the development of pulmonary vascular disease, pulmonary artery SMCs were transfected with either the full-length coding sequence of endothelial nitric oxide synthase (NOS) under the control of the CMV enhancer/promoter or with the control vector (pcDNA3.1) and injected simultaneously with the pulmonary endothelial toxin monocrotaline. At 28 d after injection the right ventricular systolic pressure was significantly decreased from 50 +/- 4 mm Hg in animals injected with the null-transfected cells to 33 +/- 3 mm Hg in animals injected with the NOS-transfected cells (P < 0.01). These results suggest that a cell-based strategy of ex vivo transfection may provide an effective nonviral approach for the selective delivery of foreign transgenes to pulmonary microvessels in the treatment of pulmonary vascular disease.