Enhanced formation of superoxide radicals has been proposed to play a major role in the development of nitrate tolerance in humans. We tested the effects of vitamin C (Vit-C) supplementation on glyceroltrinitrate (GTN)-induced hemodynamic effects during 3-d nonintermittent transdermal administration of GTN (0.4 mg/h) in nine healthy subjects. Tolerance development was monitored by changes in arterial pressure, dicrotic digital pulse pressure, and heart rate. Studies with GTN, Vit-C, or GTN/Vit-C were successively carried out at random in three different series in the same subjects. GTN treatment caused an immediate rise in arterial conductivity (a/b ratio of dicrotic pulse), but within 2 d of initiating GTN, the a/b ratio progressively decreased and reached basal levels. In addition, there was a progressive loss of the orthostatic decrease in blood pressure. However, coadministration of Vit-C and GTN fully maintained the GTN-induced changes in the orthostatic blood pressure, and the rise of a/b ratio was augmented by 310% for the duration of the test period. Changes in vascular tolerance in GTN-treated subjects were paralleled by upregulation of the activity of isolated platelets, which was also reversed by Vit-C administration. These findings demonstrate that dietary supplementation with Vit-C eliminates vascular tolerance and concomitant upregulation of ex vivo-washed platelet activity during long-term nonintermittent administration of GTN in humans.