OBJECTIVE The authors addressed three questions concerning the circadian rhythm of aqueous humor protein concentration in rabbits. First, is there an endogenous oscillator for this circadian rhythm? Second, does a circadian rhythm occur for individual aqueous humor protein components? Third, what is the role of ocular sympathetic nerves, which are more active in the dark phase, in this circadian rhythm? METHODS Adult New Zealand albino rabbits were entrained to a daily 12-hour light/12-hour dark cycle. Under a constant dark environment for 24 hours, rabbits were killed at 4-hour intervals, beginning at 2 hours before the onset of the subjective light phase. Eight rabbits were used for each of the six time points. Aqueous humor and vitreous humor were collected, and their protein concentrations were determined. Major aqueous humor protein components were resolved by polyacrylamide gel electrophoresis (PAGE), stained with silver reagent, and analyzed using densitometry. Another group of eight light-dark-entrained rabbits underwent unilateral transection of the cervical sympathetic trunk. Three weeks after the operation; the circadian elevation of intraocular pressure (IOP) at 2 hours into the dark phase was determined for both eyes. Rabbits were later killed at this time point, and total protein concentrations in aqueous humor and vitreous humor were determined in both eyes. Major aqueous humor protein components in both eyes were resolved by PAGE and were compared. RESULTS In light-dark-entrained rabbits, a circadian rhythm of protein concentration appeared in the aqueous humor under a constant dark environment. Total protein concentration in aqueous humor increased sharply in the early subjective light phase, remained relatively high during the remainder of the subjective light phase, and decreased in the subjective dark phase. Analyses of albumin and other abundant proteins in the aqueous humor showed that all of them varied similarly in a circadian pattern. In contrast, total protein concentration in the vitreous humor remained unchanged. In rabbits with unilaterally decentralized ocular sympathetic nerves, total protein concentrations in the aqueous humor and the vitreous humor in the early dark phase showed no difference between the two eyes. In addition, there was no difference in individual aqueous humor protein concentration between the two eyes. However, the nocturnal IOP elevation in the decentralized eye was less than that in the contralateral, intact eye. CONCLUSIONS The circadian rhythm of aqueous humor protein concentration in rabbits can continue without an external signal of dark-light change, indicating the existence of an endogenous oscillator. A similar circadian rhythm occurs for various major aqueous humor protein components. The nocturnal increase in ocular sympathetic activities plays a limited role in the circadian rhythm of aqueous humor protein concentration.