We have examined the influence of endothelial cell removal upon the O-methylation of the isomers of isoproterenol in the isolated rabbit aorta. Endothelial cells were removed by mechanically abrading isolated segments of rabbit aorta. The latter procedure resulted in the abolition of acetylcholine-mediated relaxation of tissues, a process dependent upon the presence of endothelial cells. In addition, histological staining and electron-microscopic analysis indicated the presence of endothelial cells in tissues not subjected to mechanical abrasion and the absence of endothelial cells in tissues subjected to abrasion. Moreover, ultrastructural analysis revealed that the influence of abrasion was limited to the luminal side of the internal elastic lamina. The removal of endothelial cells from the isolated rabbit aorta was associated with a decrease (approximately 30%) in the O-methylation of the isomers of isoproterenol. Inhibition of the extraneuronal uptake process with deoxycorticosterone (DOCA) produced a predicted decrease in the O-methylation of isoproterenol in tissues with the endothelium intact. In the absence of endothelial cells, the inhibition of the O-methylation of isoproterenol mediated by DOCA persisted. The results suggest that the O-methylation of catecholamines can occur in both medial and endothelial structures in this blood vessel and support the results of immunohistochemical studies that suggested a presence of catechol-O-methyltransferase in vascular endothelial structures. In addition, the findings exclude a possibility that the extraneuronal uptake process for catecholamines resides exclusively in endothelial structures. The functional consequences of vascular endothelial cell O-methylation are discussed.