The effect of timolol, acetazolamide, and the combination of the two drugs on the rate of aqueous formation in 18 healthy human subjects was measured during the day and at night in a placebo-controlled, double-masked, randomized study. In the absence of any drugs, aqueous flow during the day was 2.61 ±0.82 (mean ± SD) μl/min and at night, 1.08±0.59, a 59 lower flow rate when compared with the daytime value (P<0.0001). When compared with these aforementioned control values, timolol alone reduced the rate of aqueous flow by 39% (P<0.0001) in awake subjects but had no statistically significant effect on the flow rate in sleeping subjects (P=0.33). Acetazolamide alone reduced aqueous flow during the day by 21% compared with the control flow rate (P=0.02) and at night by 24% below the nocturnal flow rate in the sleeping eye (P=0.04). The combination of the two drugs reduced flow during the day by an additional 13% (P=0.024) compared with the flow rate achieved by timolol alone, and by an additional 32% (P<0.0001) compared with the flow rate reduction attained by acetazolamide alone. There was no statistically significant difference in the nocturnal flow rates achieved by acetazolamide alone or in combination with timolol (P=0.37). These data confirm previous studies demonstrating the effect of timolol, acetazolamide, and sleep on the rate of aqueous humor formation. Unlike a previous study, it was found that acetazolamide lowers the aqueous flow below the already low nocturnal flow rate that occurs spontaneously in the sleeping eye. It appears that the mechanisms which mediate the effects of sleep, timolol, and acetazolamide on aqueous formation are all different and that in the case of timolol a state of wakefulness is required for a measurable effect to occur.