The aim of the present study was to determine whether the adventitia of large arteries modulates vascular function. We developed a method to obtain functional vascular rings devoid of adventitia. Carotid and iliac arteries from 3-mo-old Sprague-Dawley rats were denuded from adventitia after treatment with collagenase followed by gentle peeling. Adventitia removal and integrity of the media was demonstrated by optical and confocal microscopy. Arterial rings with or without adventitia and with or without endothelium were mounted in an organ bath for isometric tension recording. Responses to 75 mM KCl or norepinephrine (0.1 nM-1 microM) were significantly reduced in segments without adventitia. Acetylcholine-induced relaxation (0.1 microM-0.1 mM) was enhanced in arteries without adventitia, whereas sodium nitroprusside-induced responses were not modified. These results demonstrate that the combination of stripping with a previous collagenase treatment allows us to obtain functional rings devoid of adventitia and that this layer plays a role in contractile capacity and in endothelium-modulated responses.