The pathogenesis of cigarette smoke-induced pulmonary hypertension is not understood. We previously reported that a single smoke exposure acutely but transiently upregulated gene expression of the vasoconstrictor/vasoproliferative agents endothelin (ET) and vascular endothelial growth factor in pulmonary arteries from rat lungs. To determine whether similar changes occurred with chronic smoke exposure, we exposed Hartley guinea pigs, an outbred strain that develops pulmonary hypertension, to smoke for 2, 4, or 12 wk. Small intrapulmonary artery branches were isolated using laser capture microdissection, and gene expression was determined by real-time polymerase chain reaction. In smoke-exposed animals, there were significantly elevated but variable increases in gene expression, with some animals demonstrating 30- to 50-fold increases. Increases in ET and vascular endothelial growth factor expression occurred early and persisted through the exposure period, whereas increases in expression of the vasodilator, endothelial nitric oxide synthase, developed more slowly. Protein levels of these mediators were also elevated by immunohistochemical staining and correlated with increases in gene expression levels. We conclude that, in some animals, cigarette smoke induces persisting and marked vascular production of mediators that control vascular muscularization and contraction/dilation. These changes may be important in the development of smoke-induced pulmonary hypertension.