The time-course of endothelial regrowth and functional recovery following polytetrafluoroethylene filament-induced endothelial denudation in vivo was studied in the left common carotid artery of the mouse. This technique does not result in any intimal hyperplasia, enabling the investigation of endothelial function without any confounding effect of intimal thickening. Endothelial coverage was assessed histologically, and functional recovery was assessed as restoration of receptor-mediated, endothelium-dependent relaxation to acetylcholine in vitro. Re-endothelialization of the carotid artery was complete within 8 days of denudation. However, relaxations to acetylcholine, which are mediated by endothelium-derived nitric oxide, were only partially restored 10 days after the procedure. At this time point, arterial responses to either phenylephrine, the receptor-independent endothelium-dependent dilator cyclopiazonic acid, or the nitric oxide donor diethylamine NONOate, were not significantly different to controls. At 25 days after denudation, acetylcholine-evoked responses remained significantly depressed compared to controls but at 90 days full recovery was observed. These data indicate that following mechanical denudation of the mouse carotid artery, although endothelial re-growth is complete within 8 days, recovery of endothelial cell function – assessed as the ability of the regenerated endothelium to mediate acetylcholine-stimulated relaxation – remains impaired for a prolonged period.