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To investigate whether the intraocular pressure (IOP) reduction and mechanism of action of timolol and latanoprost change between 1 and 6 weeks of treatment.Thirty participants on no ocular medications completed this double-masked, 6-visit, crossover study. At each visit IOP was determined by pneumatonometry, aqueous flow by fluorophotometry, and outflow facility by fluorophotometry and tonography. Separate values of uveoscleral outflow were calculated using the Goldmann equation, an episcleral venous pressure of 11 mm Hg, and each of the 2 outflow facility values. In a randomized fashion, both eyes were treated for 6 weeks with latanoprost 0.005% once daily or timolol 0.5% twice daily. Measurements were repeated at 1 and 6 weeks of dosing. After 6 weeks of washout, the second drug was administered in a crossover manner. One and 6 weeks of treatment were compared with appropriate baselines using 1-way analyses of variance (ANOVA).Timolol reduced aqueous flow by 27% at week 1 (P<0.001) and 16% at week 6 (P=0.03). Latanoprost increased uveoscleral outflow several fold at each visit (P<0.05). Neither drug altered outflow facility. Neither drug showed a detectable change in aqueous humor dynamics at week 6 compared with week 1. Both drugs significantly (P<0.001) reduced IOP at 1 and 6 weeks of treatment.Timolol and latanoprost significantly reduce IOP by different mechanisms. Timolol reduces aqueous flow whereas latanoprost increases uveoscleral outflow. Continued treatment with timolol or latanoprost for 6 weeks did not alter effects on aqueous humor dynamics. Outflow facility changes sometimes reported with prostaglandin analogues were not detected in this study.