Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding NG-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.