Context: The endocannabinoid (eCB) system is involved in the regulation of food intake and of peripheral metabolism. While the cross-talk between energy metabolism and the circadian system is well documented, little is known about a potential circadian modulation of human eCB activity. Objective: To define the 24-hour profile of circulating levels of the most abundant endogenous ligand of the CB1 receptor, 2-arachidonoylglycerol (2-AG), in healthy young non-obese adults studied under controlled bedtime, dietary, and activity conditions. Methods: Fourteen subjects participated in this 4-day laboratory study with fixed light-dark cycles, standardized meals and bedtimes. Sleep was recorded each night. On the third day, blood sampling at 15- to 30-min intervals began at 21:30 and continued for 24 hours. Cortisol, leptin, and ghrelin were assayed on all samples, while levels of 2-AG and of its structural analog 2-oleoylglycerol (2-OG) were measured at 60-min intervals. Results: All participants exhibited a large circadian variation of 2-AG serum concentrations with a nadir around mid-sleep, coincident with the middle of the overnight fast. Levels of 2-AG increased continually across the morning, peaking in the early to midafternoon. Peak values represented, on average, a nearly threefold increase above nocturnal nadir levels. Concentrations of 2-OG followed a similar pattern, although with a shorter morning increase and lower amplitude. Conclusions: The findings demonstrate that activity of the eCB system is profoundly modulated by circadian rhythmicity and suggest that its impact on the regulation of food intake is suppressed during sleep and maximal during early to midafternoon.