The administration of mesenchymal stem cells (MSCs) has been proposed for the treatment of pulmonary hypertension. However, the effect of intratracheally administered MSCs on the pulmonary vascular bed in monocrotaline-treated rats has not been determined. In the present study, the effect of intratracheal administration of rat MSCs (rMSCs) on monocrotaline-induced pulmonary hypertension and impaired endothelium-dependent responses were investigated in the rat. Intravenous injection of monocrotaline increased pulmonary arterial pressure and vascular resistance and decreased pulmonary vascular responses to acetylcholine without altering responses to sodium nitroprusside and without altering systemic responses to the vasodilator agents when responses were evaluated at 5 wk. The intratracheal injection of 3 x 10(6) rMSCs 2 wk after administration of monocrotaline attenuated the rise in pulmonary arterial pressure and pulmonary vascular resistance and restored pulmonary responses to acetylcholine toward values measured in control rats. Treatment with rMSCs decreased the right ventricular hypertrophy induced by monocrotaline. Immunohistochemical studies showed widespread distribution of lacZ-labeled rMSCs in lung parenchyma surrounding airways in monocrotaline-treated rats. Immunofluorescence studies revealed that transplanted rMSCs retained expression of von Willebrand factor and smooth muscle actin markers specific for endothelial and smooth muscle phenotypes. However, immunolabeled cells were not detected in the wall of pulmonary vessels. These data suggest that the decrease in pulmonary vascular resistance and improvement in response to acetylcholine an endothelium-dependent vasodilator in monocrotaline-treated rats may result from a paracrine effect of the transplanted rMSCs in lung parenchyma, which improves vascular endothelial function in the monocrotaline-injured lung.