Primary culture of pulmonary arterial smooth muscle cells is used extensively for in vitro studies of the physiology and pathophysiology of numerous lung diseases, including chronic hypoxic pulmonary hypertension (CHPH). Despite the potentially important functions of pulmonary veins in CHPH, primary culturing of pulmonary venous smooth muscle cells (PVSMCs) has received very little attention to date. No efficient and widely accepted methods have been established. Consequently, related studies have been delayed, which inhibits progress in exploring the mechanisms of CHPH and other lung diseases. In this study, we describe a simple and efficient method of obtaining primary cultures of PVSMCs isolated from rat distal pulmonary veins. By following four steps, isolation of pulmonary veins, enzymatic digestion, concentration of resuspended pellets and incubation, we acquired purified PVSMCs (>95%). PVSMCs were characterized by morphological activity and by immunoblotting and immunofluorescence staining for alpha-smooth muscle actin. Furthermore, the response of cells to 60 mM KCl was tested, confirming the presence of functional L-type voltage-dependent Ca(2+) channels that are characteristic of smooth muscle cells. In conclusion, we have established a simple and reliable technique to isolate and culture PVSMCs from rat distal pulmonary veins. These PVSMCs exhibit features consistent with vascular smooth muscle cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary vein.