Impairment of outflow facility in glaucoma causes large intraocular pressure (IOP) fluctuations that have been shown to be a risk factor for disease progression. The water-drinking provocative test (WDT) has been proposed as an indirect measurement of outflow facility to compare intraocular pressure responses of glaucoma eyes to different drugs. This study was a double-masked, randomized, parallel-group clinical trial comparing the IOP fluctuations in response to the WDT in patients using latanoprost versus unoprostone. After completing a wash-out of ocular hypotensive medications, patients with primary openangle glaucoma or ocular hypertension were randomized to receive either latanoprost (N=40) or unoprostone (N=42). IOP was measured before treatment and at 8 weeks after treatment (baseline IOP for WDT), followed by the WDT. IOP fluctuations and maximum IOP after water ingestion were compared between the two groups. Analysis of covariance was used to adjust for the effects of baseline IOP and treatment efficacy. The mean percentage reduction of IOP was 27% in patients using latanoprost, as compared to 13% in patients using unoprostone (p<0.001). Patients on treatment with latanoprost had significantly less IOP fluctuations in response to the WDT, compared to patients using unoprostone. From an overall baseline IOP of 20.0 mmHg and an overall treatment efficacy of 20%, the mean+/-standard error of the mean (SEM) of the IOP fluctuation during the WDT was 5.3+/-0.4 mmHg in the unoprostone group, and 3.6+/-0.4 mmHg in the latanoprost group (p=0.005, ANCOVA). This could represent an additional benefit of latanoprost over unoprostone in controlling the intraocular pressure of glaucomatous patients.