OBJECTIVE To evaluate changes in intraocular pressure and pupil size in glaucomatous dogs after instillation of 0.004% travoprost once in the morning, or once in the evening, or twice daily in 5-day multiple dose studies. METHODS Applanation tonometry (IOP) and pupil size (PS) measurements were obtained at 8 a.m., 10 a.m., 12 noon, 2 p.m. and 4 p.m. in eight glaucoma dogs. Methylcellulose (0.5% as placebo) was instilled in the control eye, and 0.004% travoprost was instilled in the opposite drug eye. Methylcellulose (0.5%) and 0.004% travoprost were instilled on the 2nd through to the 5th day with instillations in the morning (8.30 a.m.), or evening (8 p.m.), or twice daily (8.30 a.m. and 8 p.m.). RESULTS The mean +/- SEM diurnal changes from baseline IOP in the control and placebo eyes in all three studies ranged from 1.2 +/- 0.3 mmHg to 3.2 +/- 0.9 mmHg. The mean +/- SEM diurnal changes from the baseline IOP after 0.004% travoprost at 8 a.m. once daily for the next 4 days were 19.0 +/- 2.7 mmHg, 24.7 +/- 2.7 mmHg, 24.9 +/- 3.1 mmHg, and 24.7 +/- 3.1 mmHg, respectively, and were significantly different from the control eye. After travoprost was instilled at 8 p.m., the mean +/- SEM baseline changes from the baseline IOP in the drug eyes were 23.5 +/- 2.2 mmHg, 24.2 +/- 2.2 mmHg, 24.5 +/- 2.3 mmHg, and 24.2 +/- 2.3 mmHg, respectively. When 0.004% travoprost was instilled twice daily, the mean +/- SEM baseline IOP changes were 27.7 +/- 2.1 mmHg, 28.1 +/- 2.1 mmHg, 28.4 +/- 2.2 mmHg, and 28.5 +/- 2.2 mmHg, respectively, and were significantly different from the control eyes. Miosis of varying duration was frequent during the three studies. CONCLUSIONS Travoprost instilled once daily (a.m. or p.m.) as well as twice daily produces significant decreases in IOP and PS in the glaucomatous Beagle.