The existence of circadian rhythms in the time of onset of acute cardiovascular events has been described previously. This report describes the circadian variation in endothelial cell products, such as nitric oxide (NO) and endothelin-1 (ET-1) levels, and endothelium-dependent and -independent vasodilation in normal males. Plasma ET-1 and NO were measured every 4h in nine subjects (20-41 years old) over a 24h period. Endothelium-dependent and -independent vascular responses were measured in the forearm skin every 4h using laser Doppler imaging after iontophoresis of increasing doses of acetylcholine (ACh) and sodium nitroprusside respectively. A statistically significant circadian variation was demonstrated for the mean ACh response (P = 0.0001, ANOVA). The peak response [in arbitrary perfusion units (AU)] occurred at 16.00 hours (8.90±1.91 AU) and the lowest response at 08.00 hours (4.57±0.66 AU). A significant circadian variation was also seen for the highest dose of sodium nitroprusside (P = 0.036, ANOVA), the peak occurred at 16.00 hours (3.97±1.80 AU), and the lowest at 04.00 hours (2.62±0.58 AU) and 08.00 hours (2.58±1.16 AU). There was a significant circadian variation in the ET-1 levels (P = 0.04) with two peaks, one at 20.00 hours (0.80±0.28pg/ml) and the other at 08.00 hours (0.84±0.15pg/ml). The lowest value occurred at 16.00 hours (0.61±0.24pg/ml). There was also a borderline trend for a circadian variation in NO levels (P = 0.06), with higher levels at 20.00 hours (15.53±8.42μmol/l), and low levels at 04.00 hours (10.87±4.70μmol/l) and 08.00 hours (9.82±3.15μmol/l). ACh responses were significantly correlated with ET-1 (r = -0.3, P = 0.02) and NO (r = 0.30, P = 0.02) levels. Our findings suggest that endothelial activity has a circadian variation with attenuation in the morning. These circadian variations in endothelial activity might play an important role in the occurrence of acute cardiovascular events at this time, which are precipitated through the interplay between ET-1, NO and vascular function.