The release of endothelium-derived relaxing factor (EDRF) was examined in the rabbit carotid artery 6 weeks after denudation with an inflated balloon catheter in vivo. A concentric, fibromuscular intimal thickening of variable width developed in all areas lined with either regenerated endothelium or modified luminal smooth muscle cells. In vitro studies showed that in vessels precontracted with serotonin, only the re-endothelialized areas could relax to the endothelium-dependent dilators methacholine, substance P and the Ca2+ ionophore A23187. Re-endothelialized areas with large concentric, fibromuscular intimal thickening (between 10 and 20 cells thick) relaxed with a similar sensitivity and maximum to methacholine compared with control areas. It is concluded that (1) newly generated endothelial cells release EDRF whilst the specialized lining smooth muscle cells present 6 weeks after injury do not, and (2) that the presence of a large fibromuscular intima does not prevent EDRF from reaching the media to cause relaxation.